U.S. patent number 9,150,899 [Application Number 13/441,091] was granted by the patent office on 2015-10-06 for identification of compounds modifying a cellular response.
This patent grant is currently assigned to 2cureX. The grantee listed for this patent is Grith Hagel, Morten Hentzer, Morten Meldal, Jens Chr. Norrild, Ole Thastrup. Invention is credited to Grith Hagel, Morten Hentzer, Morten Meldal, Jens Chr. Norrild, Ole Thastrup.
United States Patent |
9,150,899 |
Thastrup , et al. |
October 6, 2015 |
Identification of compounds modifying a cellular response
Abstract
The present invention relates to methods for identifying
compounds capable of modulating a cellular response. The methods
involve attaching living cells to solid supports comprising a
library of test compounds. The test compounds are linked to the
solid support via cleavable linkers and may thus be released from
the solid supports. Solid supports comprising cells, wherein the
cellular response of interest has been modulated are selected and
the test compound of the solid support can then be identified. The
cellular response may for example be changes in complex formation
between proteins.
Inventors: |
Thastrup; Ole (Birkerod,
DK), Meldal; Morten (Copenhagen Nv, DK),
Hagel; Grith (Dragor, DK), Norrild; Jens Chr.
(Birkerod, DK), Hentzer; Morten (Holb.ae butted.k,
DK) |
Applicant: |
Name |
City |
State |
Country |
Type |
Thastrup; Ole
Meldal; Morten
Hagel; Grith
Norrild; Jens Chr.
Hentzer; Morten |
Birkerod
Copenhagen Nv
Dragor
Birkerod
Holb.ae butted.k |
N/A
N/A
N/A
N/A
N/A |
DK
DK
DK
DK
DK |
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Assignee: |
2cureX (Birkerod,
DK)
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Family
ID: |
34968405 |
Appl.
No.: |
13/441,091 |
Filed: |
April 6, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130123140 A1 |
May 16, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11569597 |
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PCT/DK2005/000347 |
May 25, 2005 |
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Foreign Application Priority Data
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May 25, 2004 [DK] |
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2004 00821 |
May 25, 2004 [DK] |
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2004 00822 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/6845 (20130101); C07K 7/08 (20130101); C07K
5/1008 (20130101); G01N 33/5023 (20130101); C07K
1/047 (20130101); C07K 7/06 (20130101); C12Q
1/025 (20130101); G01N 33/54313 (20130101); G01N
2035/00158 (20130101); C40B 30/06 (20130101); G01N
2500/10 (20130101) |
Current International
Class: |
C40B
30/06 (20060101); G01N 33/543 (20060101); G01N
33/68 (20060101); C07K 1/04 (20060101); C07K
5/103 (20060101); C07K 7/06 (20060101); C12Q
1/02 (20060101); C07K 7/08 (20060101); G01N
33/50 (20060101); G01N 35/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-2003/038431 |
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May 2003 |
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WO |
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WO-2005/045430 |
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May 2005 |
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WO |
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Other References
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HT1080 Cells, Interacts With a Cyclic RGD Peptide; The Journal of
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Analysis Using Internally Quenched Fluorescent Peptides; Biochem.
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SSR, 38(6): 53-7. cited by applicant .
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2149-54, Jul. 20, 1963. cited by applicant .
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Micro-carriers in Homogeneous Culture, Nature,16: 64-65, Oct. 7,
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Redistribution assay using a cAMP-dependent protein kinase-green
fluorescent protein chimer, Cellular Signalling, 16: 907-920, 2004.
cited by applicant .
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Antagonist, from Screening a Fully Encoded Differential Release
Combinatorial Chemical Library, Bioorganic & Medicinal
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Harpur, A. et al., Imaging FRET between spectrally similar GFP
molecules in single cells, Nature Biotechnology, 19: 167-9, Feb.
2001. cited by applicant .
Lam, K. et al., Synthesis and Screening of "One-Bead One-Compound"
Combinatorial Peptide Libraries, Methods in Enzymology, 369:
298-322, 2003. cited by applicant .
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a High-Throughput Green Fluorescent Protein-Based Recombinant Cell
Bioassay, Biochemistry, 41: 861-8, 2002. cited by applicant .
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library method to identify peptide ligands for .alpha.4.beta.1
integrin receptor in non-Hodgkin's lymphoma, Letters in Peptide
Science, 8: 171-8, 2002. cited by applicant .
Seluanov, A., DNA end joining becomes less efficient and more
error-prone during cellular senescence, PNAS, 101(20): 7624-9, May
18, 2004. cited by applicant .
Meldal, M., The One-Bead Two-Compound Assay for Solid Phase
Screening of Combinatorial Libraries, Biopolymers (Peptide
Science), 66: 93-100, 2002. cited by applicant .
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DNA ligation assays, Nature Biotechnology, 20(5): 473-7, May 2002.
cited by applicant .
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cells, Genome Research, 13(10): 2341-47, Oct. 2003. cited by
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cells, Genome Research, 13(10): 2341-47, Oct. 2003, supplemental
information 1. cited by applicant .
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cells, Genome Research, 13(10): 2341-47, Oct. 2003, supplemental
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cells, Genome Research, 13(10): 2341-47, Oct. 2003, supplemental
information 3. cited by applicant.
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Primary Examiner: Boesen; Christian
Attorney, Agent or Firm: Gifford, Krass, Sprinkle, Anderson
& Citkowski, P.C.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 11/569,597, filed Nov. 27, 2006, which is the U.S. national
stage application of PCT/DK2005/000347, filed May 25, 2005, which
claims priority to Denmark application Nos. PA200400821 and
PA200400822, both filed May 25, 2004. The entire content of all of
which is incorporated herein by reference.
Claims
The invention claimed is:
1. A method of identifying a compound modifying in the range of 1
to 5 cellular responses, wherein each cellular response is linked
to a reporter system generating a detectable output, wherein if
there is more than one cellular response, then each reporter system
is different, and wherein the cellular response is selected from
the group consisting of: i. change in morphology; ii. change in
viability; said method comprising the steps of: (a) Providing
multiple solid supports capable of supporting adherence and growth
of human primary cells, wherein the solid supports are
compartmentalized mini-reaction vessels and wherein each
compartmentalized mini-reaction vessel is linked to multiple copies
of a member of a library of test compounds via cleavable linkers
and wherein at least two solid supports comprise different library
members; and (b) Attaching human primary cells endogenously
comprising said reporter system(s) onto said solid support, and (c)
Releasing a proportion of said library member from the solid
support; and (d) Screening said solid supports for solid supports
comprising cells meeting at least one predetermined selection
criterion, wherein said selection criterion is linked directly or
indirectly to said detectable output; and (e) Selecting solid
supports comprising cells meeting said at least one selection
criterion; and (f) Identifying said library member, thereby
identifying a compound modifying in the range of 1 to 5 cellular
response.
2. The method according to claim 1, wherein said cellular response
is a change in viability.
3. The method according to claim 1, wherein said cellular response
is change in morphology.
4. The method according to claim 1, wherein said cells are primary
cells from neoplastic tissues.
5. The method according to claim 1, wherein the library is selected
from the group consisting of peptides, glycopeptides, lipopeptides,
nucleic acids (DNA or RNA), oligosaccharides; chemically modified
peptides, oligomers of amino acids glycopeptides, and small organic
molecules.
6. The method according to claim 1, wherein at least one cleavable
linker is selected from the group consisting of acid-labile,
base-labile, fluoride-labile and photo-labile linkers.
7. The method according to claim 1, wherein step c) comprises
releasing in the range of 5 to 95% of the copies of the library
members.
Description
FIELD OF INVENTION
The present invention relates to a method and tools for extracting
information relating to an influence, for example on intracellular
molecule(s), in particular an influence caused by contacting a
cellular molecule with a substance, which has been released from a
solid support to which a cell expressing the cellular component is
attached. In particular the method related to a solid support that
allow chemical synthesis of individual substances on beads of the
solid support
The method of the invention may be used as a very efficient
procedure for testing or discovering the influence of a library of
substances on a physiological process, for example in connection
with screening for new drugs, testing of substances for toxicity,
identifying drug targets for known or novel drugs. Other valuable
uses of the method and technology of the invention will be apparent
to the skilled person on the basis of the following disclosure
BACKGROUND OF INVENTION
Combinatorial synthesis of peptide as well as small-molecule
libraries has proven very useful as a method for generating vast
numbers of highly diverse compounds (see for example Comprehensive
Survey of Combinatorial Library Synthesis: 2002 Roland E. Dolle J.
Comb. Chem., 2003, pp. 693-753). To fully exploit this high
capacity of combinatorial chemistry to produce huge numbers of
compounds several technologies have been developed that allow
screening directly on the solid support (M. Meldal, 1994, METHODS:
A companion to methods of enzymology 6:417-424). In the field of
drug discovery such methods have successfully been applied for
example for the identification of enzyme modulators. The library
can be synthesized on resin beads that each carry one specific
compound, and these "one-bead-one compound" libraries are then
screened against the purified biological component of interest
(e.g. cellular proteins or peptides),
Before progressing active compounds, identified though such
procedure, further in the drug discovery process, the compound will
have to be re-synthesized and tested for efficacy in a cell-based
or in-vivo test system.
Novel ways to screen combinatorial libraries in a physiological
more correct way are assumed to greatly accelerate the drug
discovery process, and show importance in areas like chemo-genomics
and chemo-proteomics.
Screening of combinatorial libraries in intact cells have been done
by capturing mammalian or yeast cells together with a limited
number of resin-beads in a "nanodroplet" (Borchart et al. Chem Biol
1997 4:961). Compounds immobilized on the resin are released
through disruption of a photo-cleavable linker and the
compound-associated effects on the intact cells are monitored.
In an alternative method the compounds are released through
acidolysis resin-beads carrying the library members area are spread
out on a lawn of mammalian cells, and the spatial localization of a
cellular response is monitored and beads in that region is
isolated, and the remaining compound is structure elucidated
Jayawickreme et al, 1998,
Combinatorial peptide Library Protocols, Ed. Shmuel Cabilly, Humana
Press, p. 107-128). WO03/038431 describes methods for screening
combinatorial bead libraries by capturing cells from body fluids.
Beads comprising a compound enabling cells to adhere to said bead
may be selected.
US2003/0059764 describes multiplexed cell analysis systems using
non-positional or positional arrays of coded carriers.
SUMMARY OF INVENTION
It is of great importance to provide new and efficient methods for
identification of compounds influencing specific cellular
processes. In particular, such methods wherein a very large
quantity of candidate compounds may be tested for a specific effect
on a cell within a relatively short period of time.
It is therefore an object of the present invention to provide very
efficient procedures for testing or discovering the influence of
compounds of a library on a physiological process in a cell. In
particular, the methods provides means for testing very large
numbers of different compounds for one or more physiological
effects within a rather short time period. This may be obtained by
attaching living cells to resin beads coupled to a test compound.
The test compounds may be released from the resin beads and thus
influence physiological processes in said cells. Said influence(s)
may be detected and beads containing cells displaying the desired
influence(s) may be selected. Once selected the compounds coupled
to the selected beads may be identified. These methods may for
example be very useful in connection with screening for new drugs,
testing of substances for toxicity, identifying drug targets for
known or novel drugs.
Accordingly, it is a first objective of the invention to provide
methods of identifying a compound modifying at least one cellular
response, wherein each cellular response is linked to different
reporter systems generating detectable outputs, said method
comprising the steps of: (a) Providing multiple resin beads capable
of supporting growth of cells, wherein each resin bead is linked to
multiple copies of a member of a library of test compounds via a
cleavable linker and wherein at least two beads comprise different
library members; and (b) Attaching cells comprising said reporter
system(s) onto said resin beads; and (c) Releasing a proportion of
said library member from the resin bead; and (d) Screening said
resin beads for beads comprising cells meeting at least one
predetermined selection criterion, wherein said selection criterion
is linked directly or indirectly to said detectable output; and (e)
Selecting beads comprising cells meeting said at least one
selection criterion; and (f) Identifying the library member
remaining linked to the selected resin bead, thereby identifying a
compound modifying said at least one cellular response.
The method involves release of a proportion of library member. The
released library member may enter into cells in the immediate
surroundings and thus influence cellular responses within said
cells. In practical terms, the cells present in the immediate
surroundings, will be the cells attached to the resin bead, from
which the library member is released. Thus resin beads comprising
cells, wherein the particular cellular response has been modified
may be selected and the library member remaining bound to said
resin beads can be identified.
The invention furthermore relates to methods of manufacturing a
compound modifying at least one cellular response, wherein said
method comprises the steps of: a) Identifying said a compound
modifying a cellular response according to the methods described
herein b) Preparing said compound by chemical synthesis c) Thereby
manufacturing said compound
The invention also relates to methods of modulating the interaction
between two cellular molecules comprising the steps of a)
Identifying a modulating interaction between two cellular molecules
according to the methods described herein b) Incubating said
compound together with cells expressing said two cellular molecules
c) Thereby modulating the interaction between the two cellular
molecules
Depending on the nature of the two cellular molecules, specific
cellular responses may be inhibited/activated. This may in
particular be interesting for use of the compounds in therapy.
The invention furthermore relates to compounds identified by the
methods disclosed herein.
DESCRIPTION OF DRAWINGS
FIG. 1A illustrates a method of identifying a resin bead comprising
a compound influencing a cellular response linked to a reporter
system generating a fluorescent output. The method involves
cultivating cells on resin beads, fixing cells. FABS calibration
using a positive and a negative control, identification and
isolation of positive hits.
FIG. 1B illustrates a method of identifying a resin bead comprising
a compound influencing a cellular response linked to a reporter
system generating a fluorescent output detectably using a plate
reader or image acquisition analysis. The method involves 1) Grow
cells on beads for 24 hrs and Fix cells in EtOH, 2) Add app. 20
beads to each well and Identify hit wells using plate reader or
image acquisition/analysis and 3) Transfer beads from hit wells to
a new 384 well plate--one bead/well and identify hit wells using
plate reader or image acquisition. If for example 500,000 beads are
screened with 20 beads/well, approx. 25,000 wells, i.e. approx. 68
plates must be screened. With a 0.1% hit rate, there will be
approx. 500 hit wells comprising approx. 10,000 beads, which
amounts to analysis of approx. 27 plates in the second round.
Alternatively, positive beads may be picked directly (preferably
without fixation) after the first identification using image
acquisition analysis. The method may for example be used for
analysis of expression of a Cre-YFP construct.
FIG. 2A illustrates a multiplexed screen involving FABS and
microscopy. The screen involves I) identification of positive hits
by FABS as displayed in FIG. 1, followed by II) a step of
microscopy identifying resin beads comprising cells with an
internal fluorescent signal.
FIG. 2B illustrates a multiplexed screen involving two FABS
analysis. The screen involves I) identification of positive hits by
FABS as displayed in FIG. 1, followed by II) a second FABS
analysis.
FIG. 3 illustrates a plasmid map of pCRE-d2EGFP
FIG. 4 illustrates examples of cleavable linkers useful with the
present invention.
FIG. 5 illustrates spectra and structure determination by accurate
mass differences from single beads
FIG. 6 illustrates structure determination by accurate mass
differences from single beads
FIG. 7 illustrates a fragmentation pathway
FIG. 8 illustrates examples of an adhesion peptide displaying bead
covered with cells (U2OS).
DEFINITIONS
Naturally occurring amino acids are named herein using either their
1-letter or 3-letter code. If nothing else is specified amino acids
may be of D or L-form. In the description (but not in the sequence
listing) 3-letter codes starting with a capital letter indicate
amino acids of L-form, whereas 3-letter codes in small letters
indicate amino acids of D-form. Three- and one-letter abbreviations
for amino acids are used according to the recommendations from
IUPAC, see for example http://www.chem.qmw.ac.uk/iupac.
The term "a" as used herein, can mean one or more, depending on the
context in which it is used.
In the present context, the term "green fluorescent protein" or
(GFP) is intended to indicate a protein which, when expressed by a
cell, emits fluorescence upon exposure to light of the correct
excitation wavelength (cf. [(Chalfie et al. 1994)]). "GFP" as used
herein means any protein or fragment thereof capable of fluorescing
when excited with appropriate radiation. This includes fluorescent
proteins that are either naturally occurring or engineered and
proteins that have been modified to be fluorescent. Naturally
occurring fluorescent proteins have been isolated from the
jellyfish, Aequorea vistoria, the sea pansy. Renilla reniformis,
Phialidium gregarium and Discosoma coral (W. W. Ward et al. (1982)
Photochem. Photobiol, 35:803-808; Levine et al. (1982) Biochem.
Physiol., 72B:77-85; Fradkov et al. (2000), FEBS Lett.
479:127-130). GFPs have also been engineered to emit different
colors and to fluoresce more intensely in mammalian organisms (U.S.
Pat. No. 5,625,048; WO 97/28261; WO 96/23810; EP0851874; U.S. Pat.
No. 6,172,188; WO01/98338).
A variety of Aequorea-related fluorescent proteins have been
engineered to have different excitation and emission spectra by
modifying the naturally occurring amino acid sequence (D. C.
Prasher et al. (1992) Gene 111:229-233; Heim et al. (1994) Proc.
Natl. Acad. Sci. USA 91: 12501-12504; U.S. Pat. No. 5,625,048; WO
96/23810 and PCT/US97/14593).
The term "living cell" is used to indicate a cell which is
considered living according to standard criteria for that
particular type of cell such as maintenance of normal membrane
potential, cell membrane integrity and energy metabolism
The terms "image processing" and "image analysis" are used to
describe a large family of digital data analysis techniques or
combination of such techniques which reduce ordered arrays of
numbers (images) to quantitative information describing those
ordered arrays of numbers. When said ordered arrays of numbers
represent measured values from a physical process, the quantitative
information derived is therefore a measure of the physical
process.
The term "fluorescent probe" is used to indicate a fluorescent
fusion polypeptide comprising a GFP or any functional part thereof
which is N- or C-terminally fused to a biologically active
polypeptide as defined herein, optionally via a peptide linker
consisting of one or more amino acid residues, where the size of
the linker peptide in itself is not critical as long as the desired
functionality of the fluorescent probe is maintained. A fluorescent
probe according to the invention is expressed in a cell and
basically mimics the physiological behaviour of the biologically
active polypeptide moiety of the fusion polypeptide.
The term "determining the fluorescence" is used to describe the
process used to monitor a change in fluorescence properties.
The term "bioluminescence" is used to describe a process where
light is produced through a chemical reaction that natively is
occurring in a biological system. For the reaction to occur at
least two chemicals are required: the one that produces the light
(called "luciferin") and the other (called "luciferase") that
catalyzes the reaction. Sometimes the luciferin and luciferase are
brought together in one single unit (called "photoprotein" an
example of the last group is aequorin.
The term "FRET" is used to describe the occurrence of Fluorescence
resonance energy transfer between a fluorophore donor and an
acceptor fluorophore. It is a distance-dependent interaction
between the electronic excited states of two fluorophores in which
excitation is transferred from a donor fluorophore to an acceptor
fluorophore without emission of a photon. The efficiency of FRET is
dependent on the inverse sixth power of the intermolecular
separation, making it useful over distances comparable with the
dimensions of biological macromolecules. Thus. FRET is an important
technique for investigating interactions between cellular molecules
for example complex formation.
The term "BRET" is used to describe a process that is related to
FRET, but differs from FRET in that donor is a bioluminescent
protein like luciferase that generates its own luminescence
emission in the presence of a substrate, and that can pass the
energy to an acceptor fluorophore. For either BRET or FRET to work,
the donor's emission spectrum must overlap the acceptor's
absorption spectrum, their transition dipoles must be in an
appropriate orientation, and the donor and acceptor must be in
close proximity (usually within 30-80 .ANG. of each other,
depending on the degree of spectral overlap).
The term "Scintillation Proximity Assay" is used to describe an
assay determining the distance between two compounds, wherein one
compound (bound to a bead) will emit light when radiation from an
isotope occurs in close proximity and the other compound is
containing a radioactive isotope.
The term "Proximity ligation" is used to describe an assay
determining the presence of a target molecule through the
convergence of two different protein-binding reagents that
specifically recognise said target molecule. Attached to each
protein-binding reagent are nucleic acid sequences that when broad
into close proximity will create a DNA reporter sequence through a
ligation reaction (see Gullberg et al. Curr Opinion Biotechnology
2003, 14:82)
The term "mammalian cell" is intended to indicate any cell of
mammalian origin. The cell may be an established cell line, many of
which are available from The American Type Culture Collection
(ATCC, Virginia, USA) or a primary cell with a limited life span
derived from a mammalian tissue, including tissues derived from a
transgenic animal, or a newly established immortal cell line
derived from a mammalian tissue including transgenic tissues, or a
hybrid cell or cell line derived by fusing different celltypes of
mammalian origin e.g. hybridoma cell lines. The cells may
optionally express one or more non-native gene products, e.g.
receptors.
The phrase "fluorescence properties" means absorption properties,
such as wavelength and extension, and spectral properties of the
emitted light, such as wavelength, fluorescence lifetime, intensity
or polarisation, or the intracellular localisation of the
fluorophore. It may thus be localised to a specific cellular
component (e.g. organelle, membrane, cytoskeleton, molecular
structure) or it may be evenly distributed throughout the cell or
parts of the cell.
The term "fixed cells" is meant to cover cells treated with a
cytological fixative such as glutaraldehyde, methanol, acetone or
formaldehyde, treatments which serve to chemically cross-link
and/or stabilize soluble and insoluble proteins within the
structure of the cell or to dehydrate cells. Once in this state,
such proteins cannot be lost from the structure of the now-dead
cell.
The term "cell line" is meant to cover a group of cells, wherein
the cells of that group are essentially genetically
indistinguishable from each other. The cells of a cell line are
thus all progeny of the same cell.
The term "comprising" should be understood in an inclusive manner.
Hence, by way of example, a composition comprising compound X, may
comprise compound X and optionally additional compounds.
The term "multiple" should be understood as "at least two".
The term "library of test compounds" should be understood as a
collection of test compounds comprising at least 2 different test
compounds.
The term "small organic molecules or compounds" refers herein to
non-oligomeric, carbon containing compounds producible by chemical
synthesis and generally having a size of less than 600 mass
units.
The term split/mix refer herein to the process of i) dividing a
bead assembly into m portions and reacting each portions with a
different building block followed by mixing the resin into one
portion providing an even distribution throughout the assembly of
beads containing said building blocks, ii) preparing (activating)
the resin for attachment of the next building block and repeating
the process n times of dividing, reacting, mixing and activating,
thus providing an exponential growth of the number (m.sup.n) of
distinct molecular entities of complexity n each attached to
separate beads.
The term "one bead-one compound library" refers to libraries
immobilised on resin beads, wherein each individual resin bead does
not comprise more than one library member in one or multiple
copies. In a particular form of such libraries each member is
represented by multiple fragments of said member obtained by ladder
synthesis encoding.
The term "one bead-two compound library" refers to libraries
immobilised on resin beads, wherein each individual resin bead does
not comprise more than one library member in one or multiple copies
and wherein each individual resin bead in addition to said library
member also comprises an adhesion compound. All beads may comprise
identical adhesion compounds.
The term "cleavable linker" is used to describe any chemical moiety
which may be used to attach any molecule to a solid support either
covalently or via complex formation and thereafter release said
molecule by the action of either acid, base, electrophiles,
nucleophiles, oxidative agents, reductive agents, metals, heat or
light.
DETAILED DESCRIPTION OF THE INVENTION
Library of Test Compounds
In the present invention, libraries of compounds are used to screen
for compounds having a desired physiological influence on a living
cell. As used herein, the term "library" means a collection of
molecular entities or test compounds, herein also designated
"library members" obtained after a series of chemical
transformation.
In preferred embodiments of the present invention the library is a
combinatorial library. Non-limiting examples of combinatorial
libraries that may be used with the present invention and methods
of producing such libraries are given in: Comprehensive Survey of
Combinatorial Library Synthesis: 1998 Roland E. Dolle and Kingsley
H. Nelson, Jr. J. Comb. Chem., 1999, pp 235-282; Comprehensive
Survey of Combinatorial Library Synthesis: 1999 Roland E. Dolle J.
Comb. Chem., 2000, pp 383-433; Comprehensive Survey of
Combinatorial Library Synthesis: 2000 Roland E. Dolle J. Comb.
Chem., 2001, pp 477-517; Comprehensive Survey of Combinatorial
Library Synthesis: 2001 Roland E. Dolle J. Comb.
Chem., 2002, pp 369-418 and Comprehensive Survey of Combinatorial
Library Synthesis: 2002 Roland E. Dolle J. Comb. Chem., 2003, pp
693-753. The skilled person will appreciate that these protocols
may be easily be adapted to specific need of a particular
embodiment of the present invention.
In one embodiment, these molecular entities can be natural
oligomers (oligomers of building blocks occurring in Nature) such
as peptides, glycopeptides, lipopeptides, nucleic acids (DNA or
RNA), or oligosaccharides. By way of example, a natural oligomer
may be any peptide consisting of naturally occurring amino acid,
even if said peptide comprises a sequence not present in nature.
The libraries may comprise different natural oligomers or the
libraries may comprise only one kind of natural oligomer, for
example the library may be a peptide library. In another
embodiment, they can be unnatural oligomers (oligomers comprising
one or more building blocks not occurring in Nature) such as
chemically modified peptides, glycopeptides, nucleic acids (DNA or
RNA), or, oligosaccharides, and the like. Said chemical
modification may for example be the use of unnatural building
blocks connected by the natural bond linking the units (for
example, a peptide amide linkage), the use of natural building
blocks with modified linking units (for example, oligoureas as
discussed in Boeijen et al, 2001, J. Org. Chem., 66: 8454-8462;
oligosulfonamides as discussed in Monnee et al, 2000, Tetrahedron
Lett., 41: 7991-95), or combinations of these (for example, statine
amides as discussed in Dolle et al, 2000, J. Comb. Chem., 2:
716-31.). Preferred unnatural oligomers include oligomers
comprising unnatural building blocks connected to each other by a
naturally occurring bond linking. Said oligomers may thus comprise
a mixture of naturally occurring and unnatural building blocks
linked to each other by naturally occurring bonds. By way of
example, the oligomer may comprise naturally occurring amino acids
and unnatural building blocks linked by peptide bonds f.x. PNA or
LNA. Thus, in one embodiment of the invention preferred oligomers
comprise modified amino acids or amino acid (mimics). Other
preferred unnatural oligomers include, for example oligoureas, poly
azatides, aromatic C--C linked oligomers and aromatic C--N linked
oligomers. Still other preferred oligomers comprise a mixture of
natural and unnatural building blocks and natural and unnatural
linking bonds. For example, the unnatural oligomer may be any of
the oligomers mentioned in recent reviews see: Graven et al., 2001,
J. Comb. Chem., 3: 441-52; St. Hilaire et al., 2000, Angew. Chem.
Int. Ed. Engl., 39: 1162-79; James, 2001, Curr. Opin. Pharmacol.,
1: 540-6; Marcaurelle et al., 2002, Curr. Opin. Chem. Biol., 6:
289-96; Breinbauer et al., 2002, Angew. Chem. Int. Ed. Engl., 41:
2879-90. The libraries of the invention may also comprise cyclic
oligomers, for example cyclic natural oligomers, such as cyclic
peptides or cyclic unnatural oligomers. In certain embodiments of
the invention, libraries of cyclic oligomers may be advantageous to
use due to the rigid structure. This may result in higher
selectively and affinity.
In yet another embodiment, the molecular entities may comprise
non-oligomeric molecules such as peptidomimetics or other small
organic molecules. Peptidomimetics are compounds that mimic the
action of a peptidic messenger, such as bicyclic thiazolidine
lactam peptidomimetics of L-proplyl-L-leucyl-glycinamide (Khalil et
al, 1999, J. Med. Chem., 42: 2977-87). In a preferred embodiment of
the invention, the library comprises or even more preferably
consists of small organic molecules. Small organic molecules are
non-oligomeric compounds of less than about 600 mass units
containing any of a variety of possible functional groups and are
the product of chemical synthesis, or isolated from nature, or
isolated from nature and then chemically modified, and include, for
example, Bayer's urea-based kinase inhibitors (Smith et al., 2001,
Bioorg. Med. Chem. Lett., 11: 2775-78). Small organic compounds may
for example be selected from the group consisting of alcohols,
ethers, carboxylic acids, aryloxy, acyloxy, thiol, alkylthio,
arylthio, heteroarylthio, sulphonyl, sulphoxy, amino, alkylamino,
dialkylamino, acylamino, diacylamino, alkoxycarbonylamino, amides,
alkyl, branched alkyl, aryl, heteroaryl, nitro, cyano, halogeno,
silyloxy, keto, heterocycles, fused ring systems, fused
heterocycles and mixtures thereof, wherein each of the
aforementioned may be substituted independently on each position
with one or more groups selected from the group consisting of --H,
--OH, --SH, halogen, carboxyl, carbonyl, alkoxy, aryloxy, acyloxy,
alkylthio, arylthio, heteroarylthio, sulphonyl, sulphoxy, amino,
alkylamino, dialkylamino, acylamino, diacylamino,
alkoxycarbonylamino, amides, alkyl, aryl, heteroaryl, nitro, cyano,
halogeno, silyloxy, keto, heterocycles, fused ring systems, and
fused heterocycles.
Non-limiting examples of small organic molecule libraries that may
be used with the present invention and methods of producing them
may for example be found in the reviews Thompson et al., 1996,
Chem. Rev., 96: 555-600; Al-Obeidi et al., 1998, Mol. Biotechnol.,
9: 205-23; Nefzi et al., 2001, Biopolymers, 60: 212-9; Dolle, 2002,
J. Comb. Chem., 4: 369-418.
The libraries according to the invention may comprise at least 20,
such as at least 100, for example at least 1000, such as at least
10,000, for example at least 100,000, such as at least 1,000,000
different test compounds. Preferably, the libraries comprises in
the range of 20 to 10.sup.7, more preferably 50 to 7,000,000, even
more preferably 100 to 5,000,000, yet more preferably 250 to
2,000,000 different compounds. In a very preferred embodiment of
the present invention the libraries comprises in the range of 1000
to 20,000 or for example in the range of 20,000 to 200,000
different test compounds.
In preferred embodiments of the invention the library comprises in
the range of 10,000 to 1,000,000 different test compounds.
Preferably, the libraries to be used with the present invention are
immobilised on resin beads. Said resin beads may be any of the
beads described herein below. At least 2, preferably at least 20,
more preferably at least 100, even more preferably at least 1000,
yet more preferably at least 10,000, for example at least 100,000,
such as at least 1,000,000 resin beads comprising different library
members, i.e. different test compounds may be used with the methods
according to the invention. Preferably, the in the range of 20 to
10.sup.7, more preferably 100 to 7,000,000, even more preferably
1000 to 5,000,000, yet more preferably 5000 to 2,000,000, even more
preferably 10,000 to 1,000,000 resin beads comprising different
library members, are used with the methods according to the
invention.
In one very preferred embodiment of the invention, each resin bead
does not comprise more than one library member in one or more
copies, i.e. each resin bead only comprises one kind of test
compound, however said test compound may be present on the resin
bead in multiple copies. Such libraries may also be designated
one-bead-one-compound libraries. Preferably, each resin beads
comprises sufficient copies of said library member in order to
exert the desired influence of cells attached to said resin bead
and in order to analyse the chemical structure of the compound.
Such libraries may be prepared by different methods, for example by
a split/mix method or by coupling individually a specific compound
to a bead. One-bead-one compound libraries offer the advantage that
once a resin bead has been selected according to the methods
described herein, the desired compound may easily be identified
(see useful methods herein below).
The libraries may in one preferred embodiment be synthesized
directly on resin beads using a split/mix method (vide infra) which
gives rise to one-bead-one-compound libraries. Split/mix methods in
general comprise the steps of: 1. Providing several pools of resin
beads 2. Performing one or more different chemical synthesis steps
on each pool of resin beads 3. Mixing pools of resin beads, thereby
obtaining a mixed pool. 4. Splitting the mixed pool of resin beads
thereby obtaining new pools. 5. Optionally repeating step 1 and
4
Alternatively steps 3 and 4 may be as follows: 3. Splitting said
pools to obtain fractions 4. Mixing fractions from different pools,
thereby obtaining new pools
One-bead-one-compound libraries may for example be prepared as
described in M. Meldal, Multiple column synthesis of quenched
solid-phase bound fluorogenic substrates for characterization of
endoprotease specificity in Methods: A Companion to Methods in
Enzymology 6:417-424, 1994 or in M. Meldal, The One-bead
Two-Compound Assay for Solid Phase Screening of Combinatorial
Libraries in Biopolymers, Peptide Science 66:93-100, 2002; or in
Combinatorial peptide library protocols, Ed. by Shmuel Cabilly,
Humana Press, 1998, p. 1-24 and 51 to 82.
In one embodiment of the invention the library may be a library of
oligomers of amino acids immobilised on resin beads, wherein said
amino acids may be naturally occurring amino acids or any other
kind of amino acid or a mixture of both. In general, oligomers of
amino acids are referred to as peptides, regardless whether they
comprise naturally occurring or not naturally occurring amino acids
or a mixture, unless it is clear from the context that the term
only cover oligomers of naturally occurring amino acids. For
example, the oligomers may comprise or consist of at least 3, such
as at least 4, for example at least 5, such as at least 6, for
example at least 7, such as at least 10, for example at least 20,
such as in the range of 3 to 5, for example in the range of 4 to
10, such as in the range of 5 to 15, for example in the range of 10
to 20 amino acids, such as in the range of 15 to 30, for example in
the range of 3 to 50, such as in the range of 3 to 25, for example
in the range of 4 to 15 amino acids. Such libraries may for example
be prepared as described in example 1 herein below.
The library of oligomers of amino acids may in one embodiment
comprise an appended sequence. Thus, every library member may share
a common sequence, i.e. the appended sequence. In addition every
library member comprises an individual sequence. The appended
sequence may be a sequence of amino acids, for example in the range
of 3 to 5, for example in the range of 4 to 10, such as in the
range of 5 to 15, for example in the range of 10 to 20 amino acids,
such as in the range of 15 to 30, for example in the range of 3 to
50, such as in the range of 3 to 25, for example in the range of 4
to 15 amino acids.
In another embodiment of the invention the library may be a
one-bead-two-compounds library. Each individual resin bead of such
a library comprises only one library member in one or more copies.
In addition each individual resin bead comprises a second compound,
such as a cell adhesion compound. The cell adhesion compound could
for example be any of the cell adhesion compounds mentioned herein
below. It is comprised within the invention that several library
resin beads, such as all library resin beads comprises identical
adhesion compound(s) in one or more copies. One-bead-two-compound
libraries may for example be prepared by a method involving the
steps of: 1. Providing resin beads comprising a plurality of
reactive groups 2. Reacting said reactive groups with two chemical
moeities comprising different and preferably orthogonal protective
groups 3. Deprotecting a subset of the reactive groups by removal
of one kind of protective groups, preferably selective removal of
one kind of protective group, 4. Attaching or synthesizing a
split/mix library of test compounds to the deprotected reactive
group 5. Deprotecting the remaining reactive groups by removal the
other kind of protective group 6. Attaching the second compound to
the deprotected reactive groups
The method may also be performed by first attaching the second
compound and then synthesizing the library. Accordingly, the steps
of the method may be performed in the following order: 1, 2, 3, 6,
5 and 4. The library of test compounds may be first synthesized and
then attached to the resin beads or it may be synthesized directly
into the resin bead. Similarly, the second compound may be first
synthesized and then attached to the resin beads or it may be
synthesized directly into the resin bead.
Preferred resin beads are described in the section "resin beads"
herein below. The reactive group may be any suitable reactive
group, preferably however, the reactive group is either a hydroxyl
group, a thiol or a primary amino group. The reactive group may
also preferably be an azido or a secondary amino group. The
protective group may be any suitable protective group known to the
person skilled in the art, such as acid labile, alkaline labile,
fluoride labile, oxidation labile, reduction labile or photolabile
protective groups, preferably the protective group is selected from
the group consisting of Fmoc, Boc, Alloc and N.sub.3. It is
preferred that the different protective groups may be removed by
different treatment, for example that if one protective group is
acid labile, then the other is not acid labile, but instead for
example alkaline labile or photo labile. In an preferred embodiment
one protective group is Fmoc and the other protective group is
Alloc or N.sub.3. Step 3 may for example be performed by a
split/mix method as described herein above, thereby generating a
one-bead-one-compound library. The second compound is preferably a
cell adhesion compound.
In yet another embodiment of the invention the library may be a
mixed compound library, wherein each individual resin bead
comprises a plurality of library members.
Selection of an appropriate library is dependent upon the specific
embodiment of the invention. For example, a totally random library
designed to contain interesting and greatly diverse compounds may
be used with the invention. An advantage of this approach is that
the outcome of the screening is not prejudiced in any specific
manner or at least less prejudiced. Since the invention permits
screening of millions of diverse compounds, for example,
immobilized on resin beads, a large number, for example in the
range of 3 to 5 million, of random molecules can be used in the
ligand library.
Alternatively, a smaller, targeted library (hundreds to thousands
of compounds) can be used, for example, starting with a known
compound or compounds, and providing numerous variations of these
known compounds for targeted screening. For example, in embodiments
of the invention wherein compounds modulating the activity of a
specific cell surface molecule, a compound known to modulate said
specific cell surface molecule may be used as starting compound for
the preparation of a targeted library. Alternatively, a smaller
targeted library of compounds mimicking a compound known to
modulate the activity of said cell surface molecule may be
prepared, for example using computer aided modelling followed by
chemical synthesis. The smaller, targeted library can also comprise
random molecules. Examples of libraries and methods of preparing
such libraries, which may useful in embodiments of the invention,
wherein the cellular response is mediated through an intracellular
signalling pathway are known to the skilled person. The library may
contain a parallel array of random modifications of one or more
test compounds. In one embodiment, the library may be formed as a
parallel array of random modifications to a known compound or
compounds. The term "parallel array" is meant to cover synthesis of
a library by subjecting a given compound to a known set of
reactions in an isolated vessel or well. Thus, the nature of a
compound in a given container or well is known. The array of test
compounds is preferably prepared directly on resin beads using
techniques known by those skilled in the art. Briefly, the resin
may be portioned into a number of vessels or wells, usually less
than 500 and the reagents added. There is in general no mixing step
and after the appropriate washing steps, subsequent reactions are
carried out by addition of additional reagents to the wells. There
is no exponential increase in the number of compounds generated and
that is equal to the number of vessels used. The compound can be
easily identified by keeping track of the reagent added to each
well.
The library may also have been prepared by parallel synthesis using
a tag to enable identification of, what chemical synthesis steps
the individual resin bead has been submitted to. This may for
example be done by IRORI or radiofrequency tag. Alternatively,
chemical synthesis steps may be performed in parallel to preparing
a polymeric tag. Identification of the tag will thus provide
knowledge of the compound.
Attachment of a label to a compound may alter the properties of
said compound. Hence, in one embodiment of the present invention,
the compounds of the library are not labelled, i.e. the compounds
are not connected to a detectable label, such as a fluorescent
component, a nucleic acid or a nucleic acid homologue such as PNA,
a dye, a probe comprising a reactive moiety or the like. In
particular it is preferred that all compounds are not connected to
the same detectable label.
The peptides used for preparation of any of the libraries mentioned
above may be oligomers of naturally occurring or not naturally
occurring amino acids or a mixture of both, preferably they are
oligomers of the 20 amino acids naturally present in proteins,
wherein said amino acids may be in either D- or L-form. It is
preferred that each peptide (or peptide mimetic) is immobilised on
a solid support, such as any of the solid supports mentioned herein
below. More preferably the solid support is resin beads and it is
preferred that each resin bead comprises only one library member in
one or more copies.
Preferably at least 2, such as at least 10, for example at least
100, such as at least 1000, for example at least 10,000 different
peptides and/or peptide mimetics are provided. Each peptide may
comprise in the range of 2 to 100 amino acids, such as in the range
of 2 to 50 amino acids, for example 2 to 25 amino acids, such as in
the range of 2 to 15 amino acids, for example 2 to 10 amino acids,
such as in the range of 3 to 8 amino acids, for example 4 to 6
amino acids,
The invention also relates to libraries prepared by any of the
methods described above. Libraries of heterocyclic compounds
obtained by cyclisation of a peptide aldehyde through an
intramolecular Pictet-Spengler reaction may also be used with the
present invention. Such libraries may for example be any of the
libraries described in Danish patent application PA 2003 00967,
which is hereby incorporated by reference.
In one embodiment of the invention the library comprises oligomers
of amino acids, such as oligomers of 3 to 10 amino acids, such as
oligomers of 3 to 6 amino acids, for example oligomers of 3 to 4
amino acids, wherein the oligomer optionally may comprise
additional moieties, such as 1 to 4, for example 1 to 3, such as 1
to 2, for example 1 additional moiety, which is not an amino acid.
The additional moiety may be an acyl moiety, such as an acyl
halide. The additional moiety is preferably situated at one end of
the oligomer, preferably at the C-terminus. The amino acids may be
either naturally occurring or not naturally occurring amino acids
or a mixture of both. They are preferably linked via peptide bonds.
In one embodiment the library comprising compounds consisting of 3
amino acids linked via peptide bonds and an additional moeity which
may be an amino acid or for example an acyl halid. Such libraries
may for example be useful for identifying compounds capable of
interacting with inhibitors of apotosis, such as ML-IAP or XIAP.
Examples of such libraries and methods of preparing such libraries
are described herein below in Examples 4, 5a, 5b and 5c. In one
embodiment libraries prepared essentially as described in examples
5b or 5c are preferred.
In one embodiment the library may comprise or essentially consist
of oligomers of amino acids, such as oligomers of in the range of 3
to 6 amino acids, for example oligomers of 4 amino acids, wherein
the amino acids are selected from the group of amino acids
mentioned in table 3, table 4, table 5 and table 6. A preferred
example of such a library is given in example 4.
Resin Beads
The library members of this invention are preferably bound to a
solid support. Preferred solid supports to be used with the present
invention are resin beads (see herein below). The solid support may
however also be a spot or region on a surface or a plated gel or a
membrane. A spot or a region is a defined area on said surface, to
which the library member is bound via a cleavable linker. One can
therefore envisage one surface comprising a plurality of spots or
regions, wherein each such spot or region is covalently attached to
only one library member in one or more copies. Said surface could
for example be a silicium wafer, a glass surface, a plastic surface
or a gel. Plastic surface may for example be prepared from
polystyrene, polycarbonate polypropylene, ethylene and/or teflon.
Gels could be prepared from for example poly acrylamid or PEGA.
In this invention however, the compounds of the library are
preferably bound to a resin bead, conferring the advantage of
compartmentalized "mini-reaction vessels" for attachment of
cells.
In general more compounds may be screened and several of the steps
in the procedure may be performed on one bead with sufficient
material. Hence, preferably, the library is bound to resin beads.
Each member of the library is a unique compound and is physically
separated in space from the other compounds in the library,
preferably, by immobilizing the library on resin beads, wherein
each bead at the most comprises one member of the library.
Depending on the mode of library synthesis, each library member may
contain, in addition, fragments of the library member. Since ease
and speed are important features of this process invention, it is
preferred that the screening step take place on the same solid
support used for synthesis of the library, and also that
identification of the members of the binding pair can take place on
the same support, such as on a single resin bead. Thus, preferred
solid supports useful in the process invention satisfy the criteria
of not only being suitable for organic synthesis, but are also
suitable for screening procedures, such as "onbead" screening as
well as suitable for attachment of cells. It is furthermore
preferred that the resin bead is suitable for "on-bead"
identification of library members as described herein below. The
resin bead may be prepared from any suitable material such as
polystyrene, polyethylene, polyacrylamide, controlled pore glass or
PEG. The resin bead could thus for example be selected from the
group consisting of Toyopearl, sepharose, sephadex, CPG, silica,
POPOP, PEGA, SPOCC, Expansin, Tentagel, Argogel, Polystyrene,
Jandagel, polydimethylacrylamide resin, Polyacrylamide resin,
kieselghur supported resins and polystyrene supported resins.
Hydrophilic supports are preferred. Examples of preferred
hydrophilic resin beads includes TentaGel (commercially available
from Rapp polymere, Tubingen, Germany), ArgoGel (commercially
available from Argonaut Technologies Inc., San Carlos, Calif.),
PEGA (commercially available from VersaMatrix, Copenhagen), POEPOP
(Renil et al., 1996, Tetrahedron Lett., 37: 6185-88; available from
Versamatrix, Copenhagen, Denmark) and SPOCC (Rademann et al, 1999,
J. Am. Chem. Soc., 121: 5459-66; available from Versamatrix,
Copenhagen, Denmark). Examples of on-bead screening attempts are
described in the following references: Chen et al., 1996, Methods
Enzymol., 267: 211-19; Leon et al., 1998, Bioorg. Med. Chem. Lett.,
8: 2997-3002; St. Hilaire et al., 1999, J. Comb. Chem., 1: 509-23;
Smith et al., 1999, J. Comb. Chem., 1: 326-32; Graven et al., 2001,
J. Comb. Chem. 3: 441-52; Park et al., 2002, Lett. Peptide Sci., 8:
171-78). TentaGel and ArgoGel are made up of polyethylene glycol
chains grafted on to a polystyrene core. However, use of these
supports in biological screening is limited by a size restriction,
and by denaturation of certain proteins, particularly enzymes.
Preferred resin beads according to the present invention are resin
beads, useful for on-bead library synthesis, screening and
identification of ligand/protein. Hence, preferred resins according
to the present invention are resin comprising polyethylene glycol.
More preferably, the resin is PolyEthyleneGlycol Acrylamide
copolymer (PEGA), Super Permeable Organic Combinatorial Chemistry
(SPOCC) resin or PolyOxyEthylene-PolyOxyPropylene (POEPOP) resin.
Another preferred resin comprises a crosslinked polyacrylamide
resin.
PEGA (PolyEthyleneGlycol Acrylamide copolymer; Meldal M., 1992,
Tetrahedron Lett., 33: 3077-80), POEPOP
(PolyOxyEthylene-PolyOxyPropylene resin; Renil et al., 1996,
Tetrahedron Lett., 37: 6185-88) and SPOCC (Super Permeable Organic
Combinatorial Chemistry resin; Rademann et al, 1999, J. Am. Chem.
Soc., 121: 5459-66) resins are made primarily of polyethylene
glycol and swell well in organic as well as aqueous solvents.
Because they have very reduced or no non-specific binding, PEGA and
SPOCC resins have been effectively used in the screening of myriad
proteins including enzymes of different classes. Furthermore, these
resins are available in different pore sizes and can allow large
proteins to enter while retaining activity. For example, PEGA6000
resins allow proteins up to 600 kDa to enter. In the Examples
below, PEGA4000 and PEGA1900 resin with a molecular weight cut off
of 200 and 90 kDa, respectively, were used for screening. In
principle, any hydrophilic support that is useful for
compartmentalized synthesis, retains the activity of the proteins,
and has minimal non-specific binding, may be used in this process
invention.
One aspect of the invention relates to a method comprising the step
of providing multiple resin beads capable of supporting growth of
cells. Preferably, all resin beads provided are capable of
supporting growth of cells. In one preferred embodiment all resin
beads are similar and each is capable of supporting growth of
cells, wherein the resin beads only differs by comprising different
library members. In embodiments of the invention wherein the resin
beads comprise a cell adhesion molecule, it is preferred that at
least 10%, more preferably at least 20%, even more preferably at
least 30%, yet more preferably at least 40%, even more preferably
at least 50%, yet more preferably at least 60%, %, even more
preferably at least 70%, yet more preferably at least 90%, even
more preferably essentially all, yet more preferably all resin
beads comprise the cell adhesion molecule as well as a library
member.
Release of Library Compounds or of Adhesion Compound
The library of test compounds is preferably linked to the resin
beads or solid supports by a cleavable linker. Hence in one
embodiment of the invention, a proportion of the library members
may be released from the resin beads, preferably by cleaving the
cleavable linker. The thus released library members may then
interact with cells in the immediate proximity, i.e. normally with
cells attached to the same bead, and it is even possible that the
library member may enter the cells and interact with intracellular
compounds. Later selection of a single bead allows elucidation of
the structure of the specific library member remaining attached to
said bead.
Preferably, "releasing a proportion of a library member" means
releasing one or more copies of the library member attached to a
solid support or resin bead. Preferably, said copies of the library
member are released by cleaving the cleavable linker. Preferably,
in the range of 5 to 95% of all copies of a library member attached
to a resin bead are released, more preferably in the range of 10 to
90%, even more preferably in the range of 20 to 80%, such as in the
range of 30 to 70%, for example in the range of 40 to 60%, such as
at least 5%, for example at least 10%, such as at least 20%, such
as at least 30%, for example at least 40%, such as at least 50%,
for example at least 60%, such as at least 70%, for example at
least 80%, such as at least 90%, for example at least 95%, such as
at the most 5%, for example at the most 10%, such as at the most
20%, such as at the most 30%, for example at the most 40%, such as
at the most 50%, for example at the most 60%, such as at the most
70%, for example at the most 80%, such as at the most 90%, for
example at the most 95% of all copies of a library member attached
to a resin bead are released.
It is also comprised within the invention that the adhesion
compound may be attached to the resin bead via a cleavable linker.
Cleavage of said cleavable linker may release the adhesion compound
as well as cells attached to said adhesion compound. When the
cleavable linkers linking the library compound and the adhesion
compound, respectively, are differentially cleavable, then
selective release of either library compound or adhesion compound
may be achieved.
The cleavable linker may be any chemical moiety which may be used
to attach any molecule to a solid support either covalently or via
complex formation, and thereafter release said molecule by the
action of either acid, base, electrophiles, nucleophiles, oxidative
agents, reductive agents, metals or light. Preferably, the
cleavable linker attaches the library member to the solid support
covalently. Depending on the nature of the cleavable linker, a
person skilled in the art will be capable of controlling cleavage
of the cleavable linker, so only a proportion of the copies of a
library member are released. A comprehensive review describing
state of the art for "cleavable linkers" is "Linkers and Cleavage
Strategies in Solid-Phase Organic Synthesis and Combinatorial
Chemistry", F. Guillier, D. Orain, and M. Bradley, Chem. Rev. 2000,
100, 2091-2157. Any of the cleavable linkers described therein may
be used with the present invention.
Examples of useful acid labile linkers include the most commonly
used linkers for acidic detachment from a solid support, the Wang
and Rink linkers (FIGS. 4A and B). Detachment of peptide esters
from Wang linkers require up to 95% TFA in DCM whereas detachment
of Rink esters (FIG. 4B, X.dbd.OH) may be cleaved under milder
conditions (AcOH-DCM 1:9) which does not cleave the normal
protection groups on the peptides. The Rink amides (FIG. 4A,
X.dbd.NH.sub.2) require 95% TFA (aq). Partial detachment of the
compounds attached to the resin may also be achieved using gaseous
acids such as HCL or TFA vapour in a sealed container. The use of
gases allow rigorous control of the degree of cleavage obtained
with concentration of acid and time of exposure. The skilled person
may readily establish a suitable concentration of acid and time of
exposure to obtain a desired degree of cleavage.
Examples of useful base-labile linkers includes Wang and HMBA
linkers, which may be cleaved under alkaline conditions.
Saponification with 0.1 M NaOH may be applied but even milder
conditions such as potassium carbonate in MeOH are applicable. The
HMBA linker is stable to TFA under normal conditions.
In a preferred embodiment the cleavable linker is a light sensitive
cleavable linker which, upon the action of light with a given wave
length and intensity, may release any active compound from the
solid support.
Photo-labile linkers provide a tool for solid phase synthesis which
enables the detachment of the synthesized molecules in the presence
of acid or base-sensitive functionalities within the molecules. In
1973 Rich proposed the use of o-nitrobenzyl type of linkers
(nitrated analogs of the Wang linker). Irradiation with UV-light
gave detachment of the free acids or amides although only in
moderate yields. Detachment yields could be improved by applying
the NBA type linkers (see FIG. 4E). Even better result have been
obtained with the Holmes-type linkers (FIG. 4F). Detachment from
photolabile linkers is performed by illuminating the resins with
ultraviolet light, preferable at 365 nm. The wave length and
intensity of the light and the time of exposure might need
optimization for the individual case. A person skilled in the art
can readily establish conditions wherein a desired proportion of
copies of a library member are released. Detachment yields may be
over 90% under ideal conditions.
Example of a preferred photo sensitive cleavable linker:
##STR00001##
Depending on the nature of the cleavable linker, the library member
may be released using different methods. For example, if the linker
is photo labile, the library member may be released by
illumination. The release should preferably be partial, so that
only a proportion of the library member is released. The person
skilled in the art will readily be able to establish the conditions
required for partial release using a specific cleavable linker. An
example of how to achieve partial release is given in example 6
herein below.
It is also comprised within the present invention that a library
member may be linked to a resin bead via different cleavable
linker. For example some copies of a library member may be linked
to a resin bead via a first kind of cleavable linker, whereas other
copies of the same library member may be linked to the resin bead
via a second kind of cleavable linker. Preferably, the first kind
of cleavable linker is cleavable by another method than the second
kind of cleavable linker. By way of example, the first cleavable
linker could be acid or base labile, whereas the second kind of
cleavable linker could be photolabile. Thus, some copies of the
library member could be released during the screening procedure of
the invention, for example during step c) in the method outlined in
the "Summary" herein above, whereas other copies could be retained
on the resin beads and released during the identification, for
example during step f) in the method outlined in the "Summary"
herein above. Thus, releasing a proportion of a library member
could be controlled by using different cleavable linkers.
Cells
The cells to be used with the present invention may be any useful
cells available or prepared for the purpose. Preferably, the cells
are selected from the group consisting of mammalian cells. For
example the cells may be human cells. The cells may be cells
capable of growing in suspension or they may be adherent cells.
Adherent cells may preferably be cultivated directly on the resin
beads used with the invention (see also herein below). It is
preferred that the cells are adherent cells. Cells with a better
adherence are preferred over cells with a poorer adherence. Cells
which adhere well to resin beads comprising an adhesion compound as
described herein above are very preferred.
Cells could for example be primary cells or established cell lines.
Preferred cell lines include but are not limited to those mentioned
in table 1.
TABLE-US-00001 TABLE 1 Cell line Species Tissue Morphology 3T3-L1
Mouse Embryonic fibroblast Fibroblast 3T3-Swiss Mouse Embryo
Fibroblast albino (CCL-92) A10 Rat thoracic aorta Myoblast Att 20
Mouse Pituitary Small round cells BAE Cow Aorta Endothelial Balb/c
Mouse Embryonic fibroblast Fibroblast BHK:R P.1#4aa PTP1B fl BHK-21
Hamster Kidney Fibroblast BHK467 Hamster Kidney BHK570 Hamster
Kidney Fibroblast BJ Human Foreskin Fibroblast C2C12 Mouse Muscle
Myoblast Caki-1 Human Kidney Epithelial CAL-54 Human Kidney
Epithelial CHOhIR Chinese Ovary Fibroblast hamster CHO-K1 Hamster
Ovary Epithelial COS 1 Monkey Kidney Fibroblast COS 7 Monkey Kidney
Fibroblast G-8 Mouse Muscle Myoblast GT1-7 HCT 116 Human Colorectal
Epithelial HEK293 Human Embryonic kidney Epithelial Hela Human
Cervix Epithelial adenocarcinoma HEP-G2 Human Liver Epithelial
HT-1080 Human Fibrosarcoma Epithelial HT-29 Human Colon Epithelial
HUVEC Human umbilical vein Endothelial Ins-1 Jurkat clone E6-1
Human T lymphocyte Lymphoblastoid K-562 Human Bone marrov
Lymphoblastoid L-6 Rat Muscle Myoblast MCF 7 Human Mammary Gland
Epithelial MDA-MB-231 Human Adenocarcinoma Epithelial MDA-MB-468
Human Mammary Gland Epithelial MDCK Canine Kidney Epithelial Min 6
Mv 1 Lu (NBL-7) Mink Lung Epithelial NIH-3T3 Mouse Embryo
Fibroblast PAE Pig Aorta PC 12 Rat Adrenal gland PC-3 Human
Prostate Epithelial RAT2 Rat Normal Fibroblast RAW 264.7 Mouse
Monocyte RIN Rat Epithelial SK-ML-28 Human Melanoma SK-N-AS Human
Neuroblastoma Epithelial SK-N-DZ Human Neuroblastoma Epithelial
SK-N-F1 Human Brain Epithelial SK-NM-C Human Neuroepithelioma
Epithelial SK-N-SH Human Caucasian Epithelial neuroblastoma SW480
Human Colorectal Epithelial U-2 OS Human Bone, osteosarcoma
Epithelial U-87 MG Human Brain Epithelial U937 Human Lymphoma
Monocyte VERO Monkey Kidney Fibroblast-like WI-38 Human Lung
Fibroblast WM-266-4 Human Skin Epithelial WEHI Human
In one embodiment of the invention the cells have been genetically
or otherwise modified in order to enhance their usability with the
present invention. The modification may be stable or only transient
or a mixture of both. For example, the cells may have been modified
to contain one or more of the reporter systems described herein
below. Depending on the nature of the reporter system this may be
achieved by a number of different methods. For example, if the
reporter system comprises a nucleic acid, said nucleic acid may be
inserted into said cell by conventional recombinant techniques (see
below).
In another preferred example the cell comprises nucleic acid
comprising first nucleotide sequences encoding cellular proteins or
polypeptides being part of an intracellular signal transduction
pathway operationally linked to a reporter system detecting the
enzymatic activity or subcellular localization of said first
sequences, or detecting direct interactions between these first
sequences.
Useful second sequences includes for example promoters active in
the particular cells, for example mammalian promoters, viral
promoters or synthetic promoters. A large number of useful
eukaryotic promoters are known to the person skilled in the art,
useful promoters are for example described in"Mechanism of
Transcription" (1998) Cold Spring Harbor Symposia on Quantitative
Biology Vol. LXIII; Cold Spring Harbor Laboratory Press
Such promoters may be constitutively active or they may be active
only temporarily. In one example the promoter may be regulated by
an external signal, for example the promoter may be inducible or
repressable.
The nucleic acid may be inserted into the cells by any useful
method, for example by conventional recombinant techniques, such as
any of the techniques described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory,
New York, USA
In another embodiment the cells are primary cells. Primary cells
are cells with a limited life span that preferably are derived from
a mammalian tissue. Preferred primary cells are cells which are
adherent. The mammalian tissue may for example be a human tissue,
such as healthy or diseased tissue. In one embodiment the tissue is
or comprises a neoplastic tissue, for example tissue removed from a
cancer patient by surgery, for example from a patient suffering
from melanoma, breast cancer or colon cancer. The tissue may also
be hypertrophic cells, such as cardiac myocytes. Preferably said
cancer patient has not been subjected to radiotherapy prior to
surgery. In embodiments of the invention wherein the cells are
primary cells it is preferred that the reporter system is
endogenous to said primary cells.
Cell Attachment to Resin Beads and Cell Cultivation
The present invention relates to methods comprising the step of
attaching cells comprising a reporter system(s) to resin beads. The
cells may for example attach to said resin beads directly or by
attaching a second compound conferring adhesion to the resin
bead.
The resin beads useful for the present invention should preferably
be able to support cell growth. The resin beads may per se be able
to support cell growth, however frequently the resin beads will
comprise a cell adhesion compound that enables the resin beads to
support growth of cells. Said cell adhesion compound may be coupled
to said resin beads by any useful means known to the person skilled
in the art depending on the nature of the cell adhesion
compound.
Any cell adhesion compound known to the person skilled in the art
may be used with the present invention. It is frequently an
advantage if the cell adhesion compound comprises at least one
positively charged moiety at neutral pH, more preferably the cell
adhesion compound has a positive overall netcharge at neutral
pH.
In one preferred embodiment of the invention the cell adhesion
compound comprises a peptide or a polypeptide, more preferably the
cell adhesion compound consists of a peptide. Such peptides are
herein also designated "adhesion peptides".
Said peptide preferably consists of in the range of 3 to 100,
preferably in the range of 3 to 75, more preferably in the range of
3 to 50, even more preferably in the range of 3 to 30, yet more
preferably in the range of 3 to 25, even more preferably in the
range of 3 to 20, yet more preferably in the range of 3 to 15, such
as in the range of 3 to 10, for example in the range of 3 to 8, for
example in the range of 6 to 7 amino acids. In general, it is
sufficient if the peptide comprises at least 3 amino acids.
It is preferred that the peptide comprises at least one amino acid
selected from the group consisting of arginine and lysine, more
preferably the peptide comprises at least 2 basic amino acids, such
as 3 basic amino acids selected from the group consisting of Arg
and Lys, even more preferably the peptide has an overall positive
netcharge. In one preferred embodiment the peptide comprises the
following sequence of 4 amino acids: basic-basic-lipophilic-basic.
Basic amino acids may for example be selected from the group
consisting of arginine and lysine, whereas the lipophilic amino
acid may be selected from the group consisting of Gly, Ala, Val,
Leu, Ile, Phe, Trp, Pro and Met of either D or L-form. Preferably,
the peptide comprise at least 1, preferably at least 2, more
preferably at least 3, even more preferably at least 4 amino acid
on the D-form, yet more preferably all amino acids are on the
D-form. Preferably D-amino acids are used to enhance the metabolic
stability but also L-amino acids may be used.
Preferred examples of peptides useful as cell adhesion compounds
are given in table 2 herein below:
TABLE-US-00002 TABLE 2 No 1 2 3 4 5 6 7 SEQ ID NO 1 ala arg ile arg
ile gln his SEQ ID: 1 2 ala lys cys arg trp cys met SEQ ID: 2 3 ala
lys ala arg cys lys ser SEQ ID: 3 4 ala lys tyr trp ser tyr lys SEQ
ID: 4 5 ala his leu tyr arg asn lys SEQ ID: 5 6 ala arg arg cys phe
arg asp SEQ ID: 6 7 ala ala arg his cys tyr tyr SEQ ID: 7 8 ala tyr
tyr cys gln gln arg SEQ ID: 8 9 ala asp leu lys arg pro met SEQ ID:
9 10 ala gly gly lys arg lys phe SEQ ID: 10 11 ala pro arg lys arg
cys gly SEQ ID: 11 12 ala thr arg arg val ala arg SEQ ID: 12 13 ala
gly lys lys asn lys asn SEQ ID: 13 14 ala ala lys arg trp lys phe
SEQ ID: 14 15 ala arg trp pro tyr arg gly SEQ ID: 15 16 ala leu tyr
trp thr trp arg SEQ ID: 16 17 ala ala tyr arg trp tyr arg SEQ ID:
17 18 ala arg cys ile arg gly asp SEQ ID: 18 19 ala thr lys cys lys
gly arg SEQ ID: 19 20 ala val tyr met arg asn ile SEQ ID: 20 21 ala
arg lys arg ile arg gln SEQ ID: 21 22 ala lys ile arg glu lys arg
SEQ ID: 22 23 ala arg arg phe lys met tyr SEQ ID. 23 24 arg arg phe
lys SEQ ID: 24 25 arg arg ile arg SEQ ID: 25 26 leu arg his arg Leu
lys SEQ ID: 26 27 lys phe gly gln lys (cys) SEQ ID: 27 28 lys val
tyr met his lys SEQ ID. 28 29 ile arg tyr arg leu arg SEQ ID: 29 30
ala gln arg pro arg trp SEQ ID: 30 trp tyr ala lys arg arg SEQ ID:
31 lys arg ile arg gln arg leu arg SEQ ID: 32 lys arg ile arg gln
arg lys SEQ ID: 33 arg ile arg gln arg SEQ ID: 34 arg gln arg ile
arg SEQ ID: 35 No 1 2 3 4 5 6 7 SEQ ID NO lys phe gly gln lys cys
SEQ ID: 36 arg arg leu leu pro ile SEQ ID: 37 pro phe arg lys lys
cys SEQ ID: 38 tyr arg trp arg ile Ala SEQ ID: 39 arg ser lys arg
ile Asn SEQ ID: 40 arg ser ala lys arg cys SEQ ID: 41 lys lys gln
phe trp Phe SEQ ID: 42 arg met lys leu his lys SEQ ID: 43 arg his
trp gly arg ile SEQ ID: 44 thr lys arg leu lys thr SEQ ID: 45 thr
lys gly lys ala lys SEQ ID: 46 ala lys thr arg his arg SEQ ID: 47
asn arg pro arg val arg SEQ ID: 48 val pro arg lys val gln SEQ ID:
49 lys met arg tyr cys gln SEQ ID: 50 ile arg lys his leu ile SEQ
ID: 51 pro arg arg val val ile SEQ ID: 52 lys arg glu ser lys arg
SEQ ID: 53 ser arg lys asp arg lys SEQ ID: 54 arg cys lys lys leu
ile SEQ ID: 55 arg lys leu arg val asn SEQ ID: 56 val arg thr val
arg val SEQ ID: 57 arg ala phe lys tyr tyr SEQ ID: 58 ile thr arg
arg thr gln SEQ ID: 59 lys met pro lys lys asn SEQ ID: 60 lys pro
lys met met cys SEQ ID: 61 lys lys met arg phe trp SEQ ID: 62 lys
lys lys phe tyr tyr SEQ ID: 63 lys ser asn lys val arg SEQ ID: 64
lys trp pro his his arg SEQ ID: 65 arg his ile gln trp tyr SEQ ID:
66 leu arg leu lys pro lys SEQ ID: 67 glu arg lys arg cys thr SEQ
ID: 68 arg arg ala arg gln asp SEQ ID: 69 arg glu lys gly ala arg
SEQ ID: 70
Furthermore, preferred peptide may be any of the peptides
identified by any of SEQ ID: 1 to 70, preferably any of SEQ ID: 1
to 23 and 26 to 35, such as SEQ ID: 1 to 23, for example SEQ ID: 25
to 35, wherein 3 amino acids, preferably 2 amino acids, more
preferably 1 amino acid have been substituted for another amino
acid. Preferably, said substition is a conservative substitution,
i.e. substition for an amino acid with similar characteristics.
Said characteristic could for example be acidic/basic properties,
polarity or lipophilicity. It is also comprised within the
invention that the peptide may be a peptide of above mentioned size
comprising any of the peptides identified by SEQ ID: 1 to 70. In
particular, in order to immoblised the peptide on a resin bead it
may be useful to synthesise the adhesion peptide on an amino acid
immobilized on the resin bead, for example a Gly.
In one embodiment the peptide is preferably selected from the group
consisting of peptides identified by SEQ ID: 21 to 23 and 36 to 35,
more preferably from the group consisting of 26 to 35, even more
preferably SEQ ID:35. In another embodiment the peptide defined by
SEQ ID:21 is preferred.
In one embodiment of the invention it is preferred that the peptide
has low or essentially no fluorescent properties. It is
particularly preferred that the peptide has low or essentially no
fluorescent properties when attached to a solid support, such as a
resin bead. By "essentially no fluorescent properties" is meant
that the peptide does not emit any detectable fluorescence. This is
in particularly relevant for embodiments of the invention wherein
the detectable output is fluorescence (see herein below). Preferred
peptides to use with this embodiment of the invention may be
selected from the group consisting of SEQ ID:26 to 35.
Peptides useful as cell adhesion compounds may be identified using
any suitable method. Said method may for example include the steps
of i) synthesizing or coupling a test peptide on to a resin bead;
ii) incubating said resin bead with cells under cell cultivation
conditions; iii) testing whether said cells attach to said resin
bead iv) identification of the peptide sequence wherein the test
peptide is useful as cell adhesion compound If more cells attach to
said resin bead in the presence, than in the absence of said test
peptide. Preferably, the test peptide is useful as cell adhesion
compound If at least 200, more preferably at least 500, even more
preferably at least 1000 cells attach to said resin bead after
incubation. This is in particular the case in embodiments of the
invention, wherein the resin beads are PEGA beads. For example
useful test peptides may be identified as described in example 1
herein below.
In embodiments of the invention wherein it is preferred that the
peptide has no or low fluorescence it is preferred that the method
comprises an additional step performed at any point subsequent to
step i), such as immediately subsequent to step i) prior to step
ii). Said additional step comprises testing whether said peptide
has fluorescent properties. This may for example be performed by
sorting resin beads in a FABS or manually with the aid of a
fluorescence microscope. If this is done prior to step ii) then
only resin beads with no or low fluorescence properties are
incubated with cells, A non-limiting example of a useful method is
described in example 2b.
The peptide may be coupled to the resin bead by any useful method,
for example by synthesising the peptide directly onto an amino
functionalised resin bead using a standard Fmoc-protocol for
peptide synthesis. Other protective groups may be used instead of
Fmoc, for example Boc. N.sub.3 or Alloc. In one embodiment Alloc is
the preferred protective group. It is preferred that different
protecting groups are used for synthesis of the adhesion peptide or
for library synthesis. The peptide may also be synthesised by
anchoring an Fmoc amino acid to a hydroxyl functionalised resin
bead, such as a hydroxymethylbenzoic acid (HMBA) derivatised PEGA
resin followed by peptide assembly using standard Fmoc technology
as described in B. Blankemeyer-Menge, M. Nimtz, and R. Frank, An
Efficient method for anchoring Fmoc-amino acids to
hydroxyl-functionalised solid supports. Tetrahedron Lett.
31:1701-1704, 1990 and A. Dryland and R. C. Sheppard. Peptide
synthesis. Part 11. A system for continuous flow solid phase
peptide synthesis using fluorenylmethoxycarbonyl-amino acid
pentafluorophenyl esters. Tetrahedron 44(3):859-876, 1988
Sidechains may be protected with acid labile protecting groups such
as t-Bu, Trt, Pmc, Boc etc. The protected peptide may for example
be cleaved off the resin using alkaline conditions or hydrazine and
the structure may be determined e.g. by on bead Edman Degradtion.
The HMBA-linked peptide may also be cleaved under mild alkaline
condition.
In one embodiment the peptide may be linked to the resin bead via a
linker, which may be a cleavable linker. This may for example be
achieved by synthesizing the linker directly on resin beads or
coupling the linker to the resin beads and subsequently coupling or
synthesizing the library onto the resin beads. Thus, before
coupling of the library the linker preferably comprises a
protective group as described herein above. The cleavable linker
may be any of the cleavable linkers described herein above. If the
resin beads are coupled to the library via a cleavable linker it is
preferred that the cleavable linker linking the adhesion compound
is differentially cleavable.
In embodiments wherein cells adhere to the resin bead via the
adhesion compound and the adhesion compound is attached to the
resin bead via a cleavable linker, cells may be at least partially
or even essentially fully released from the resin bead by cleavage
of the cleavable linker.
Testing whether cells attach to resin beads may be done by any
conventional methods, such as by manual inspection with the aid of
a light microscope. If the cells have fluorescent properties, for
example if the cells express a fluorescent protein, then resin
beads with attached cells may be identified using a fluorescent
microscope or a FABS, preferably a fluorescent microscope.
In one preferred embodiment of the invention, the cells may be
cultivated directly on the resin beads. In general, a method of
cultivating cells on resin beads may comprise the steps of
Providing resin beads capable of supporting growth of cells Seeding
cells onto said resin bead Incubating said resin beads comprising
said cells in a cell culture medium under cell cultivation
conditions Optionally allowing said cells to divide on said resin
bead Thereby cultivating cells on resin beads
The cells may adhere actively to the resin beads and will then
generally be referred to as adherent cells.
Cells cultivation conditions depends on the specific cells. For a
large number of mammalian cells, such conditions comprise high
humidity, preferably close to 100%, approximately 5% CO.sub.2 and
around 37.degree. C. It is often desirable to keep the resin beads
immersed in a suitable cultivation medium and frequently it is also
desirable that the resin beads can be circulated within said
medium, for example by stirring or rotation. Said stirring or
rotation may be continuous or in intervals. It is also possible
that the container comprising the resin beads is simply rocked
gently a few times every now and then.
In one embodiment of the invention more than one cell line or type
of primary cell is attached to or cultivated on the beads. Hence
for example 2, such as 3, for example 4, such as 5, for example 6,
such as 7, for example 8, such as 9, for example 10, such as in the
range of 10 to 20, for example in the range of 20 to 50, such as
more than 50 different cell lines may be attached to or cultivated
on said beads. Also different specific primary cells may be
attached to the cultivated beads.
It is possible that a subgroup of resin beads only comprise one
cell line or a specific kind of primary cells and another subgroup
of resin beads comprises another cell line or another specific kind
of primary cell and so forth. However, it is also possible that in
principle every resin beads comprises all the different cell lines
and/or different primary cells.
Intermediates between these two extremes may also be envisaged.
Preferably, said different cell lines and/or primary cells comprise
different reporter systems, hence it is possible that the different
cell lines are derived from the same parent cell lined by insertion
of different reporter systems. However, the different cell lines
and/or primary cells may also be unrelated.
Cellular Molecules
In one particularly preferred embodiment of the invention the
methods of the invention involve identification of compounds
modulating a cellular response, which is mediated through an
interaction between cellular molecules, more preferably through an
interaction between intracellular molecules. Cellular molecules may
be any cellular molecule, such as proteins, polypeptides, DNA, RNA,
molecules of non-protein nature or metal-ions. In preferred
embodiment, the cellular molecule is a protein or polypeptide.
Intracellular molecules are molecules that are not accessible from
the extracellular surface of intact cells. Intracellular molecules
may for example be proteins, polypeptides, DNA. RNA, molecules of
non-protein nature or metal-ions. In one preferred embodiment,
intracellular molecules are proteins or polypeptides not accessible
from the extracellular surface of intact cells.
In one embodiment, the cellular response is mediated through an
interaction between cellular molecules of a cellular signal
transduction pathway. Hence, the invention, for example may be
useful for identifying compounds modulating the activity of a
signal transduction pathway. Such compounds could for example
activate or repress a signal transduction pathways by modulating
the interaction between different or the same cellular molecules,
modulating the catalytic activity of enzymes, modulating the
synthesis or degradation of cellular molecules, modulating
transcriptional activity, modulating the localization or movement
of cellular molecules. modulating the level of cellular molecules
(i.e. in a specific cellular compartment or on average throughout
the whole cell)
Within the context of the present invention the term "signal
transduction pathway" should be understood in its common cell
biological meaning, i.e. modulation of an intracellular event
triggered by a cell surface receptor.
Signal transduction pathways may for example involve steps of
changed catalytic activity of enzymes, phosphorylation, cleavage of
proteins, synthesis of cAMP, activation of transcription,
inhibition of transcription, change i intracellular Ca.sup.2+
concentration, change in membrane potential, subcellular
relocalisation of cellular components, complex formation of
cellular components, degradation of cellular components and/or
change in energy metabolism
The signal transduction pathway could for example be a pathway
activated/repressed by a cell surface receptor selected from the
group consisting of G-protein coupled receptors (GPCR), protein
kinase coupled receptors, receptor kinases with intrinsic kinase
activity, orphan receptors or transmembrane channels. The signal
transduction pathway may also be a pathway resulting in modulation
of transcription, for example modulation of transcription regulated
by a response element, for example a response element selected from
the group consisting of CRE, SRE, TRE and AP-1 In one embodiment of
the invention the signal transduction pathway is a pathway
resulting in apoptosis.
Preferably the cellular molecules are proteins or parts thereof or
derivatives thereof, more preferably the cellular molecules are
proteins. Even more preferably the cellular molecules belong to the
classes of: serine/threonine protein kinases; tyrosine protein
kinases, protein phosphatases, phospholipid dependent
serine/threonine protein kinases, calmodulin dependent
serine/threonine protein kinases, mitogenactivated serine/threonine
protein kinases, cycline dependent serine/threonine protein
kinases, transcription factors, structural proteins, protein
scaffolds, proteases, such as caspases, metallo-matrix-proteases,
rennin, cathepsins, viral proteases, secretases or ADAM family
proteases, or hydrolases, nucleases, synthases, isomerases,
polymerises, oxido-reductases, ATPases or GTPases.
The cellular molecules are more preferably proteins that are known
to participate in protein-protein interactions or complex
formations. Such proteins can be selected from proteins listed in
databases like BIND (Biomolecular Interaction Network Database)
http://bind.ca.
In one embodiment of the invention the cellular molecules are
involved in regulation of apoptosis. Thus the cellular molecules
may be proteins or functional fragments thereof involved in
regulation of apoptosis. Proteins involved in apoptosis includes
for example caspases, inhibitors of apoptosis (IAPs) or Smac.
Inhibitors of apoptosis may for example be XIAP, cIAP1/BIRC2,
ML-IAP/BIRC7, DIAP1, DIAP2, OPIAP3, cIAP2, NAIP, Apollon or
Survivin (see also Vaux and Silke, 2005, Nature Reviews,
6:287-297). Thus, in one example proteins involved in
protein-protein interaction may be Smac and XIAP or ML-IAP or a
Smac binding fragment thereof. Smac binding fragments of XIAP
preferably comprises the BIR3 domain of XIAP, whereas Smac binding
fragments of ML-IAP preferably comprises the BIR domain. The domain
structure of IAPs is well described, see for example Wu, G., Chai,
J., Suber, T. L., Wu, J.-W., Du, C., Wang, X., and Shi, Y. (2000)
Nature 408, 1008-1012; Matthew C. Franklin et al., Biochemistry
2003, 42, 8223-8231; and Liston et al. Oncogene. 2003 Nov. 24;
22(53):8568-80
A cell surface receptor within the meaning of the present invention
is preferably a protein, more preferably a protein that is
accessible from the extracellular surface. Yet more preferably, the
cell surface molecule is a cell surface protein receptor (herein
also merely designated "receptor"). A "receptor" within the meaning
of the present invention, is a molecule, which at least sometimes
is localised at the cell surface and which is capable or
associating with at least one ligand. The ligand binding site is
accessible from the extracellular surface. Frequently, association
with said ligand may alter the activity of the receptor.
Cellular Response
The invention relates to methods of identifying compounds
modulating, such as activating or inhibiting, a cellular response
linked to a reporter system. The reporter system may be any of the
reporter systems described herein below. The methods disclosed by
the present invention may be used to identify compounds modifying
any cellular response, which is or may be linked to a reporter
system generating a detectable output. The person skilled in the
art will appreciate that the specific methods disclosed herein may
be adapted to any such cellular response. Below, non-limiting
examples of cellular responses are described.
In a particularly preferred embodiment of the invention, the
cellular response is mediated through interaction between cellular
molecules, such as intracellular molecules. The cellular molecules
may for example be components of a signal transduction pathway, and
thus the cellular response may be activation or repression of a
signal transduction pathway. Hence, the cellular response may for
example be modulation of a signal transduction pathway within a
cell, such as modulation of a signal transduction pathway mediated
by a cell surface molecule. By "activation of a receptor" is meant
that the receptor is influenced in a manner that it activates
downstream signalling events. Accordingly, the methods according to
the present invention may be employed to identify activators or
inhibitors of signal transduction pathways.
Examples of modulations of signal transduction pathway includes:
Upregulation or downregulation of the level of a member of the
pathway Relocalisation of a member of the pathway Complex formation
between members of the pathway or between members of the pathway
with other cellular compounds Enhanced or reduced transcription
from genes regulated by the pathway Modification by for example
phosphorylation of a member of the pathway Activation or inhibition
of an enzyme of the pathway Degradation of a cellular compounds due
to upregulation or downregulation of the pathway Altered secretion
of a compound Change in ion-flux Morphological changes Change in
viability
In a preferred embodiment the modulation of a signal transduction
pathway can for example be monitored by measuring: the enzymatic
activity of an enzyme being part of said signal transduction
pathway the level of cyclic nucleotides, i.e. cAMP or cGMP the
activity of transcription factors the level of specific proteins as
quantified through standard proteomics techniques the level of
inositol or lipid phosphates the level of phosphorylation of
specific proteins as quantified through standard proteomics
techniques the binding between two or more proteins or polypeptides
the cellular localization of proteins or polypeptides
The enzymatic activity could for example be the enzymatic activity
of serine/threonine protein kinases or of tyrosine protein kinases
or of protein phosphatases or of phospholipid dependent
serine/threonine protein kinases or of calmodulin dependent
serine/threonine protein kinases or of mitogenactivated
serine/threonine protein kinases or of cycline dependent
serine/threonine protein kinases or of proteases or of hydrolases
or of nucleases or of synthases or of isomerases or of polymerises
or of oxido-reductases or of ATPases or of GTPases.
The cellular response may in one embodiment be modulation of
transcriptional activity, such as activation or reduction of
transcription of one or more genes. In particular, activation or
reduction of transcription of genes regulated by a response
element. Said response element could for example be selected from
the group consisting of CRE, SRE, TRE and AP-1.
Hence, the cellular response may also be an increased or decreased
level of a particular mRNA within a cell.
By the term "regulated by a response element" is meant that
transcription is modulated by said response element, however other
elements may also modulate transcription of said gene. By the term
"activation of response element" is meant increased transcription
of genes regulated by said response element.
In another embodiment of the invention the cellular response is:
change in the intracellular level of a compound; or change in the
level of a compound within a specific cellular compartment, for
example within the cytoplasm, in the golgi, in the endoplasmatic
reticulum, in lysosomes, in endosomes or in the nucleus
The compound may be any compound, preferably a naturally occurring
compound. Frequently, the compound is a compound endogenous to the
cell. The compound may thus for example be a salt, an ion, a
nucleotide or a derivative thereof, a peptide, a saccharide, a
lipid or a biomacromolecule. Biomacromolecules includes for example
RNA such as mRNA, polypeptides and proteins. An example of an ion
is Ca.sup.2+ and an example of a nucleotide derivative is cAMP.
In yet another embodiment of the invention the cellular response is
relocalisation of a compound. Relocalisation may for example be
concentration of a compound otherwise dispersed in one or more
specific locations relocalisation from one cellular compartment to
another, for example relocalisation from the cellular membrane to
the cytoplasma. relocalisation from one location within a
compartment to another location within the same compartment
internalisation of an extracellular compound
The compound may be any compound, such as any of the compounds
mentioned in the section above. In one preferred embodiment the
compound, which is relocalised is a biomacromolecule, such as RNA,
polypeptides or proteins. For example, the compound may be a cell
surface receptor (receptor). The cellular response may thus be
internalisation of said receptor or relocalisation of said receptor
from the cellular membrane to the cytoplasma.
In one embodiment of the invention the cellular response is change
in the activity of a compound, such as an increase or a decrease in
the activity of a compound. Said compound may for example be an
enzyme. The cellular response may for example be induction of the
activity of a caspase. Preferred caspases are Caspase 3 or 7.
In another embodiment of the invention the cellular response is
change in phosphorylation of a compound.
In another embodiment of the invention the cellular response is
formation or disruption of a complex between compounds.
In another embodiment of the invention the cellular response is
change in the concentration of a compound.
The cellular response may also be altered secretion of a compound,
such as increased or decreased secretion of a compound. Said
compound could for example be a biomacromolecule, such as a
protein, a polypeptide, a peptide, a hormone, a cytokine, or the
like.
In another embodiment of the invention the cellular response is
change in pH in an intracellular compartment, for example in the
cytoplasm.
In yet another embodiment the cellular response is a change in a
membrane potential, for example a change in membrane potential over
the cell membrane or over the mitochondria membrane.
In an even further embodiment of the invention the cellular
response is change in morphology, such as change in size or shape.
The cellular response may also be change in viability (e.g.
apoptosis or necrosis), such as change in viability under specific
conditions.
In a preferred embodiment of the present invention the cellular
response is change in interaction between two or more cellular
molecules, preferably between two cellular molecules, such as
establishment of an interaction between two or more cellular
molecules or disruption of an interaction between two or more
cellular molecules. Thus the cellular response may be formation of
a complex or disruption of a complex. The cellular molecules may be
any of the cellular molecules mentioned above, however, preferably
the cellular molecules are proteins or fragments thereof.
In one embodiment of the present invention the cellular response is
induction or facilitation of apoptosis in living cells, such as
induction or facilitation of apoptosis in tumour cells, preferably
induction of apoptosis. The cellular response may also be induction
or facilitation of apoptosis in cells that have undergone an
apoptosis promoting treatment. The cellular response may also be
induction or facilitation of apoptosis in cells that have undergone
an apoptosis inhibiting treatment, In another embodiment of the
invention the cellular response is inhibition or reduction of
apoptosis, for example reduction of apoptosis in cells prone to
undergo apoptosis or reduction in apoptosis in cells that have
undergone an apoptosis promoting treatment.
The apoptosis promoting treatment may be contacting cells with an
inducer of apoptosis. The inducer of apopotosis may be any compound
known to be capable of inducing apoptosis, for example the compound
may be staurosporine (STS). Alternatively, the apoptosis promoting
treatment may be illumination with radiation, such as with UV-light
with a predetermined wave length and intensity.
The methods according to the invention may also include
identification of compounds modulating more than one cellular
response, such as 2, for example 3, such as 4, for example 5, such
as more than 5 different cellular responses. Said cellular
responses may be any of the responses discussed above.
Reporter System
The reporter system to be used with the present invention should be
selected according to the particular cellular response. The
reporter system should be capable of generating a detectable
output.
In some embodiments of the invention the reporter system may be
identical to the cellular response. This is in particular true when
the cellular response may be detected without the aid of an
additional reporter system, for example when the cellular response
is an increase/decrease in the level of a compound, relocalisation
of a compound, change in membrane potential, change in pH, change
in morphology or the like.
Hence, the reporter system may be a system endogenous to said
cells. For example, the reporter system may comprise the endogenous
system regulating the intracellular level of an endogenous
compound. By way of example, the reporter system may be the
endogenous system of a cell regulating the intracellular Ca.sup.2+
level.
In another example, the reporter system comprises the intracellular
localisation of an endogenous compound.
In yet another example, the reporter system may comprise the
activity of an enzyme. This may in particular be relevant when the
cellular response is modulation of an enzymatic activity or
modulation of a signal transduction pathway which modulates an
enzymatic activity. Then the reporter system could be direct
detection of for example the enzymatic activity of serine/threonine
protein kinases or of tyrosine protein kinases or of protein
phosphatases or of phospholipid dependent serine/threonine protein
kinases or of calmodulin dependent serine/threonine protein kinases
or of mitogenactivated serine/threonine protein kinases or of
cycline dependent serine/threonine protein kinases or of proteases,
such as caspases, metallo-matrix-proteases, rennin, cathepsins,
viral proteases, secretases or ADAM family proteases or of
hydrolases or of nucleases or of synthases or of isomerases or of
polymerises or of oxido-reductases or of ATPases or of GTPases.
It is preferred that said enzymatic activity may be detected for
example because the enzymatic activity leads to formation of a
coloured compound, a fluorescent compound, a radioactive compound
or the like. This may be achieve by the use of appropriate
substrates. If the enzyme is a protease, the enzymatic activity may
for example be detected by use of a substrate, which generates a
detectable output when cleaved by said protease. For example the
substrate could be a peptide or a polypeptide comprising a
fluorescent moeity and a quencher, wherein cleavage would lead to
formation of two peptides/polypeptides, wherein one comprises the
fluorescent moeity and the other comprises the quencher.
Fluorescence would thus be detectable only in the presence of an
active protease.
In embodiments wherein the enzyme is a caspase, the reporter system
may be a substrate for said caspase. Useful caspase substrates are
known to the skilled person and several caspase substrates are
commercially available, for example from Beckman Coulter Inc.
Examples of Caspase substrates are Caspase 3 and/or 7 substrates.
It is preferred that cleavage of the substrates is readily
detectable. Thus fluorogenic substrates comprising a quenching
group which may be cleaved of by caspases may be useful. Cleavage
of such substrate can simply be detected by determining
fluorescence. Non-limiting examples of fluorogenic caspase
substrates are the CellProbe HT Caspase 3/7 Whole Cell Assay,
Beckman Coulter, Inc, or any of the substrates described in U.S.
Pat. No. 6,342,611. It is not required that such a reporter system
is introduced permanently into living cells. Thus the substrate may
be added directly to the cell culture medium or to an assay buffer
comprising resin beads with cells. Thus such a reporter system may
also be useful in embodiments of the invention wherein primary
cells are employed. A non-limiting example of a useful Caspase
assay is given in example 12 herein below.
However, the reporter system may also be heterologous to the cell,
i.e. a reporter system which has been inserted into the cell for
example by recombinant techniques.
In several embodiments of the invention the reporter system
comprises a fusion protein comprising a first protein and
detectable polypeptide. The detectable polypeptide may for example
be an enzyme or part thereof, a transcription factor or part
thereof or a bioluminiscent protein, such as a fluorescent protein.
Preferred detectable polypeptides are luciferase or fluorescent
proteins. The first protein may be selected according to the
cellular response. If the cellular response is change in level or
location of a given protein, the first protein could be that
particular protein. If the cellular response is change in
interaction between two or more proteins, the first protein could
be one of the proteins taking part in that interaction.
In embodiments of the invention, wherein the cellular response is
modulation of transcription from gene(s) regulated by a response
element, it is preferred that the report system comprises a nucleic
acid comprising a nucleotide sequence encoding a detectable
polypeptide operably linked to a response element, the activity of
which is modulated by the cellular response.
In embodiments of the invention, wherein the cellular response is
modulation of a signal transduction pathway, the reporter system
may comprises a nucleic acid comprising a nucleotide sequence
encoding a detectable polypeptide operably linked to a response
element, the activity of which is modulated by said signal
transduction pathway.
For example, if the cellular response is modulation of a signal
transduction pathway influencing the activity of CRE and/or SRE,
then the reporter system may comprise a nucleic acid comprising a
nucleotide sequence encoding a detectable polypeptide operably
linked to a response element selected from the group consisting of
cAMP response element (CRE) and serum response element (SRE).
Examples of such signal transduction pathways include the signal
transduction pathways modulated by GPCR of the rhodopsin family or
secretin family and by protein kinase receptors and receptors
belonging to the family of receptor kinases.
By way of example: 1) If the cellular response is activation of a
signal transduction pathway activated by a GPCR coupled to a
G.sub.S (see herein above) that stimulates adenylate cyclase, then
the reporter system may be a nucleic acid comprising a nucleotide
sequence encoding a detectable polypeptide operably linked to CRE.
Activation of said GPCR may then be detected by detection of
increased levels of said detectable polypeptide. 2) If the cellular
response is activation of signal transduction pathway activated by
a GPCR coupled to a G.sub.I (see herein above) that inhibits
adenylate cyclase, then the reporter system may be a nucleic acid
comprising a nucleotide sequence encoding a detectable polypeptide
operably linked to CRE. Activation of said GPCR may then be
detected by detection of decreased levels of said detectable
polypeptide.
Similarly, if the cellular response is modulation of a signal
transduction pathway that influences the activity of TRE, then the
reporter system may comprise a nucleic acid comprising a nucleotide
sequence encoding a detectable polypeptide operably linked to TPA
response element (TRE). Examples are GPCRs that are linked to
activation of Protein Kinase C such as Gq coupled receptors (see
herein above).
Similarly, if the cellular response is modulation of a signal
transduction pathway that influences the activity of SRE, then the
reporter system may comprise a nucleic acid comprising a nucleotide
sequence encoding a detectable polypeptide operably linked to SRE.
Examples of such signal transduction pathways include the signal
transduction pathways modulated by growth hormones or cytokines
through protein kinase receptors and receptors belonging to the
family of receptor kinases.
Similarly, if the cellular response is modulation of a signal
transduction pathway that influences the activity of AP-1, then the
reporter system may comprise a nucleic acid comprising a nucleotide
sequence encoding a detectable polypeptide operably linked to AP-1.
Examples of such signal transduction pathways include the signal
transduction pathways modulated by cytokines or growth factors
cytokines through protein kinase receptors and receptors belonging
to the family of receptor kinases
The detectable polypeptide may be any detectable polypeptide,
however preferably the detectable polypeptide is selected from the
group consisting of fluorescent proteins and enzymes.
Fluorescent proteins may for example be green fluorescent protein
(GFP) and fluorescent mutants thereof, such as yellow fluorescent
protein (YFP) or cyan fluorescent protein (CFP). The fluorescent
protein can also be a protein complex, e.g. a di- or tetramer of a
fluorescent protein, such as dsRed. Enzymes may for example be
selected from the group consisting of luciferase, CAT,
galactosidase, alkaline phosphatase and beta-lactamase.
In one embodiment of the invention the reporter system may comprise
a bioluminescent moiety. For example, if the cellular response is
relocalisation of a compound, then the reporter system may for
example be said compound linked to a luminescent moiety, such as a
fluorescent moeity. Hence, for example if the cellular response is
relocalisation of a polypeptide the reporter system may be a
chimeric protein made up of said polypeptide and a fluorescent
protein, such as GFP, YFP or CFP. In one preferred embodiment said
polypeptide may be receptor.
In one embodiment of the invention the reporter system may detect
the level of a cellular molecule, such as a protein. This may for
example be achieved by quantifying the amount of a compound i.e. an
antibody that specifically binds to the cellular molecule. The
quantification can for example be achieved by covalently coupling a
fluorescent, bioluminescent or coloured moiety to said compound.
The quantification could be confined to a specific cellular
compartment.
In one embodiment of the invention the reporter system may detect
the level of modification of a cellular molecule for example but
not limited to phosphorylation, glycosylation or ubiquitination.
This may for example be achieved by quantifying the amount of a
compound i.e. an antibody that specifically binds to the modified
cellular molecule. The quantification can for example be achieved
by covalently coupling a fluorescent, bioluminescent or coloured
moiety to said compound. The quantification could be confined to a
specific cellular compartment.
In one embodiment of the invention the reporter system may detect
complex formation or disruption between two cellular proteins
(designated first protein and second protein in this paragraph).
This is in particular relevant when the cellular response is change
in interaction between two cellular proteins. Several different
reporter systems may be used to detect interaction between a first
and a second protein. Below preferred reporter systems are
described, however, the invention is not limited to these specific
reporter systems.
The reporter system may for example comprise the first protein
linked to a bioluminescent moiety, such as luciferase and the other
protein linked to a fluorescent moiety, such as a fluorescent
protein. Such reporter systems are referred to as "BRET reporter
systems" herein. The bioluminescent moeity should preferably be
able to directly or indirectly generate light of a wavelength
capable of exciting the fluorescent moiety. The skilled person will
readily be able to select useful bioluminescent moeities and
fluorescent moeities. Preferably, the BRET reporter system
comprises a first chimeric protein comprising the first protein
linked to a bioluminescent protein, preferably luciferase and a
second chimeric protein comprising the second protein linked to a
fluorescent protein. Such a reporter system may be introduced into
a cell by introducing nucleic acids encoding the first and the
second chimeric proteins under control of suitable promoters into
said cell. Direct interaction between the proteins can after
expression of the two chimeric proteins be detected through
occurrence of BRET (Bioluminescence Resonance Energy Transfer). In
one embodiment, BRET2 technology may be used which is based on
energy transfer between a bioluminescent donor (a Renilla
luciferase (Rluc) fusion protein) and a fluorescent acceptor (a
Green Fluorescent Protein (GFP2) fusion protein). In presence of
its substrate DeepBlueC.TM. (a coelenterazine derivative). Rluc
emits blue light (.about.395 nm). Thus the reporter system may
comprise a first chimeric protein comprising the first protein and
Rluc and a second chimeric protein comprising the second protein
and GFP2. A protein-protein interaction between Rluc and GFP2
chimeric proteins allows energy transfer to GFP2, which reemits
green light (510 nm). Expression of Rluc alone, in the presence of
the substrate DeepBlueC.TM., gives an emission spectrum with a peak
at .about.395 nm, whereas when the Rluc and GFP2 chimeric proteins
interact, there is efficient energy transfer between Rluc and GFP2
and the 510 nm signal represents a major peak.
In another similar reporter system the first protein and the second
protein are linked to different fluorescent moieties, preferably a
fluorescent proteins. Such reporter systems are referred to as
"FRET reporter systems" herein. Preferably, one fluorescent moiety
is capable of emitting light of a wavelength capable of exciting
the other fluorescent moeity. FRET reporter systems preferably
comprise a first chimeric protein comprising the first protein and
a fluorescent protein and a second chimeric protein comprising the
second protein and another fluorescent protein. It is then possible
to detect the complex formation through the occurrence of FRET
(Fluorescence Resonance Energy Transfer). BRET or FRET according to
the present invention may for example be performed as described in
(Nicolas B, R Jockers, and T Issad Trends in Pharmacological
Sciences 23 (8):351-354, 2002; and/or A. Roda, M. Guardigli, P.
Pasini, and M. Mirasoli. Anal. Bioanal. Chem 377 (5):826-833,
2003)
Complex formation may also be detected by proximity ligation. In
such an embodiment the reporter system comprises two affinity
probes raised against the first and the second protein. Such
reporter systems are designated "proximity ligation reporter
systems" herein. When the two proteins come in close proximity a
ligation reaction creates a DNA reporter sequence that can be
amplified. The amplified sequence can be detected by any useful
method, for example it may be detected through photolabelling.
Preferably, the DNA reporter sequence is amplified by PCR, rolling
circle replication or ligation chain reaction. In order to detect
the amplified sequence, the sequence may for example be amplified
using primers labelled with a detectable label, such as a
fluorescent label or the sequence may be detected using a
detectably labelled probe, such as a fluorescently labelled probe.
The affinity probes in general comprise or consist of a binding
moeity and a nucleic acid moeity. The binding moiety of the
affinity probes can be any molecule that binds either the first or
the second protein with high affinity. Preferably, the binding
moeity is capable of specifically recognising and binding either
the first or the second protein. Examples of useful binding
moieties of affinity probes are monoclonal- or polyclonal
antibodies or antigen binding fragments thereof, chimeric
antibodies, recombinant antibodies, single chain antibodies or
aptamers. Antibodies may be prepared using any conventional method
known to the person skilled in the art. Aptamers may be prepared by
any method known to the skilled person, for example by iterative
cycles of screening nucleic acid libraries for compounds capable of
binding a tare nucleic acid molecules selected for their ability to
specifically bind a target. Aptamers may for example be produced
using a SELEX process (Sun S Curr Opin Mol Ther 2000 Feb. 2:100-5;
Jayasena S D Clin Chem 1999 September 45:1628-50). The nucleic acid
moeity may comprise or consist of any nucleic acid sequence,
preferably a sequence, which when ligated to another nucleic acid
moeity creates a DNA reporter sequence, which can be amplified by
PCR, rolling cycle amplification or ligase chain reaction using
appropriate primers. A person skilled in the art can design useful
nucleic acid moeity sequences and corresponding primers. The
affinity probes can be introduced into cells by a number of
different methods, for example they may be introduced into the
cells after said cells have been fixed and permeabilized or they
can be introduced by using traditional cDNA transfection methods,
for example by using standard procedure for Fugene6 transfection.
Proximity ligation may for example be carried out as described in
Frederiksson et al. Nature Biotechnology 2002, 20: 473; Gullberg et
al. Curr Opinion Biotechnology 2003, 14: 82. The reporter system
may also be a "two-hybrid reporter system". Two-hybrid reporter
systems comprises two chimeric proteins, wherein the first chimeric
protein comprises the first protein fused to a DNA binding domain
and the second chimeric protein comprises the second protein fused
to a transactivating domain. Furthermore, the two hybrid reporter
system comprises a reporter construct comprising a nucleic acid
sequence encoding a detectable polypeptide the expression of which
is controlled by the transactivating/DNA binding domain. Thus if
the first protein interacts with the second protein the DNA binding
domain and the transactivating domain are brought into close
proximity and may activate transcription from the reporter
construct. Interaction can then be determined by detection of the
detectable polypeptide. The detectable polypeptide may be any of
the detectable polypeptides mentioned herein above. Two-hybrid
reporter systems are well described in the art, see for example
U.S. Pat. No. 5,283,173.
The reporter system may also be an enzyme complementation reporter
system. Enzyme complementation reporter systems comprises two
chimeric proteins, wherein the first chimeric protein comprises the
first protein fused to a first part of an enzyme and the second
chimeric protein comprises the second protein fused to a second
part of an enzyme. The first and the second part of an enzyme
should together constitute a functional enzyme. Thus, when the
first protein interacts with the second protein the first part and
the second part of an enzyme will form a functional enzyme, the
activity of which may be determined.
One example of an enzyme useful for enzyme complementation system
is DHFR (dihydrofolate reductase), where the activity of the
reconstituted enzyme is monitored as a fluorescent read-out based
on stoichiometric binding of fluorescein-methotrexate to
reconstituted DHFR (Remy I, Michnick S W Proc Natl Acad Sci USA
1999 May 96:5394-9).
Hence, if the cellular response is relocalisation of a cell surface
molecule, then the reporter system may comprise a fluorescent
moiety covalently coupled to said cell surface molecule.
In some embodiments of the invention the cellular response is
modulation of a signal transduction pathway involving activation of
phospholipase C. Phospholipase C may for example be activated by
GPCRs coupled to G.sub.Q (see herein above). Activation of
phospholipase C in general leads to increase in the intracellular
level of Ca.sup.2+ and thus in such embodiments the reporter system
may be the intracellular Ca.sup.2+ level. This reporter system may
thus be endogenous to the cell.
When the cellular response is induction/facilitation of apoptosis a
number of reporter systems may be employed. Induction/facilitation
of apoptosis may be determined by determining caspase activity as
described herein above. Induction/facilitation of apoptosis may
also be determined by determining cell growth/number of cells, for
example cell growth/number of cells after cultivation of for
example normal cells or tumour cells, such as cells expressing high
levels of XIAP or ML-IAP. Other methods of determining apoptosis
are well known to the skilled person.
Detectable Output
The detectable output may be any output, which is detectable
directly or indirectly. For example the detectable output may be
the concentration of a compound within a cell, localisation of a
compound within a cell, luminiscense, activity of an enzyme or the
like.
In preferred embodiments of the invention the detectable output is
luminiscense, fluorescence, bioluminescence, FRET or BRET.
Bioluminiscence may be detected by any conventional methods, for
example with the aid of a Plate reader. BRET or FRET may for
example be detected using FABS, a plate reader, a fluorescence
microscope or the like.
Alternatively, the detectable output may preferably be linked
(directly or indirectly) to a bioluminescent signal.
However, the detectable output could also be radioactivity, a
coloured compound or a colour signal, a heavy metal, an electrical
potential, a redox potential, a temperature or the detectable
output may be linked to a radioactive signal, a coloured compound
or a colour signal or a heavy metal or an electrical potential, or
a redox potential or a temperature. Said radioactive signal could
for example be .sup.35S, .sup.32P, .sup.3H. The coloured compound
could for example be the product of any of the enzymatic reaction
described herein elsewhere. The heavy metal could for example be
gold.
In embodiments of the invention, wherein the cellular response is
change in the intracellular level of a compound or change in the
level of a compound within a specific cellular compartment, then
the detectable output may be said level of said compound. Depending
on the nature of the compound, said level may be detected directly
or indirectly.
If the compound for example is a fluorescent compound, the level of
said compound may be determined by determining the fluorescence
properties. This may be done by any suitable means, for example by
the aid of a fluorescence microscope, a FACS (Fluorescence
Activated Cell Sorter), a FABS (Fluorescence Activated Bead
Sorter), fluorescence plate-reader or a fluorescence
spectrometer,
If the compound for example is an enzyme then the level of said
compound may be determined by determining the activity of said
enzyme. By way of example, if the enzyme catalyses a reaction
leading to a product, which is directly detectable, for example by
colorimetric or chemiluminescent detection techniques, the activity
of said enzyme may be detected by detecting said compound. For
example, if the enzyme is luciferase, the activity of said enzyme
may be detected by detecting emission of light upon oxidation of
the added substrate, luciferin.
Several other enzymes such as CAT, .beta.-galactosidase, alkaline
phosphatase, horseradish peroxidase and beta-lactamase are, when
provided with suitable substrates, capable of catalysing reactions
leading to coloured or chemiluminescent products, which may be
detected using any colorimetric or chemiluminescent detection
technique.
If the compound for example is Ca.sup.2+, then the intracellular
concentration of said ion can be measured by using any suitable
method, for example by inserting into the cells Ca.sup.2+ binding
fluorescent compounds like Fura-2, Fluo-3 or Fluo-4 (Molecular
Probes), which change fluorescent properties according to a changed
Ca.sup.2+ concentration. A non-limiting example of a method of
determining cytosolic free Ca.sup.2+ is given in example 13 herein
below. Other ion concentrations can be monitored using suitable
fluorescent compounds, which for example are available from
Molecular Probes Inc.
If the compound for example is a protein, then it may for example
be detected using a first specific binding partner. Said first
specific binding partner could be a second protein capable of
specifically interacting with said protein, such as a specific
antibody or said first specific binding partner could be an
aptamer. Said first specific binding partner could be conjugated to
a directly detectable compound, such as a fluorescent compound, a
radioactive compound or a heavy metal or to an indirectly
detectable compound, such as an enzyme, which for example could be
any of the enzymes mentioned herein above. It is also possible that
the first specific binding partner may be detected with a second
specific binding partner, capable of interacting specifically with
the first specific binding partner. Said second specific binding
partner may be conjugated to a directly or indirectly detectable
compound similarly to the first specific binding partner.
Additional specific binding partners may be used.
In embodiments of the invention wherein the cellular response is
relocalisation of a compound the detectable output could be a
detectable label conjugated to said compound. In particular, the
compound may be conjugated to a directly detectable label, such as
a fluorescent label or a heavy metal. Thus the localisation of the
compound may be directly detected, for example using a fluorescence
microscope. Fluorescent plate-reader, fluorescence spectrometer, a
FACS or a FABS instrument In one preferred embodiment the compound
is a fusion protein comprising a protein of interest and a
fluorescent protein, such as GFP. The compound may thus be a
fluorescent probe. Thus the detectable output may be localisation
of a fluorescent signal. Alternatively, the compound is a fusion
protein comprising the protein of interest and a tag. Said tag
could be a tag specifically interacting with a specific binding
partner, for example the tag could be an HA-tag or a flash domain.
Alternatively, localisation of a compound may be determined with
the aid of a specific binding partner as outlined above.
Intracellular localisation may also be detected using methods
capable of detecting distance between two compounds, for example
BRET or FRET.
In embodiments of the invention wherein the cellular response is
change of activity of a compound, the detectable output may be a
product of said activity. I.e. when said compound is an enzyme the
detectable output could be a product of a reaction catalysed by
said enzyme. Said product could thus be a coloured product or a
chemiluminiscent product as discussed herein above.
In embodiments of the invention wherein the cellular response is
enhanced or reduced transcription from one or more genes, then the
cellular response could be mRNA transcribed from said gene, a
protein encoded by said gene or in case the protein is an enzyme,
the detectable output could be a product of a reaction catalysed by
said enzyme. The enzyme and the products could be any of the
enzymes or products discussed herein above.
mRNA may be detected by any useful means, for example with the aid
of a probe capable of hybridising specifically with said mRNA. Said
probe could be labelled with a directly detectable label, for
example a radioactive compound, a fluorescent compound or a heavy
metal or an indirectly detectable label such as an enzyme or a
specific binding partner.
Said protein may be detected with the aid of specific binding
partners as outlined herein above. However, in a preferred
embodiment the protein is a fluorescent protein and may thus be
detected directly. Hence, the detectable output could be
bioluminescence, such as fluorescence.
In embodiments of the invention wherein the cellular response is
modification by for example phosphorylation of a compound this can
be detected through binding of an antibody that specifically bind
the phosphorylated protein said antibody can then be quantified by
specific fluorescence labelling.
In embodiments of the invention wherein the cellular response is
change in pH in an intracellular compartment, the detectable output
will in general be said pH. The pH may be determined using any
suitable method, for example using a pH indicator or a pH-meter.
For example the pH may be determined using a fluorescent indicator
for intracellular pH. Suitable compounds are compounds with a
fluorescent excitation profile which is pH-dependent, such as BCECF
(available from Molecular Probes).
In embodiments of the invention wherein the cellular response is a
change in a membrane potential, the detectable output will in
general be said membrane potential. The membrane potential may be
determined using any suitable method such as applying a fluorescent
molecule to cells that distribute over the membrane dependent upon
the membrane potential. Examples of such compounds are DiBAC,
various ANEP dyes, JC-1 and JC-9 (Molecular Probes). For example,
JC-1 and JC-9 are cationic dyes that exhibit potential-dependent
accumulation in mitochondria leading to a shift in fluorescence
emission from green to red. Thus mitochondrial depolarization may
for example be determined by decrease in red/green fluorescence
intensity ratio (see also product information from Molecular
Probes). ANEP dyes are in particularly useful for detection of
changes in membrane potential. The fluorescence can be read for
instance by a fluorescence microscope, a fluorescence plate-reader,
a FACS, or a FABS instrument.
In embodiment of the invention wherein the cellular response is
change in morphology, the detectable output will in general be the
morphology of the cell. The morphology may be observed using any
suitable method for example by the aid of a microscope, using a
FACS or FABS.
In embodiments of the invention where the cellular response is
change in an interaction between two cellular proteins, the
detectable outout may for example be BRET or FRET, which is
detectable by determining the occurrence of fluorescence of a given
wavelength. BRET or FRET may for example be detected using a FABS.
FACS, fluorescent microscope or any other equipment useful for
detection of fluorescence.
In embodiments of the invention wherein the reporter system is
proximity ligation the detectable output is dependent on the
detectable label used to label the amplified DNA reporter sequence.
In embodiments, wherein the DNA reporter sequence is amplified
using fluorescently labelled primers, the detectable output will be
said fluorescent label, which may be detected using a FABS. FACS,
fluorescent microscope or any other equipment useful for detection
of fluorescence.
Depending on the detectable output, it will frequently be an
advantage to fix cells prior to detecting said detectable output.
However, in some embodiments of the invention it is preferred that
the cells are not fixed. Cells may be fixed according to any useful
protocol (see also definitions herein above)
Selection
The methods according to the invention involves screening resin
beads for beads comprising cells meeting at least one predetermined
selection criterion, wherein said selection criterion is linked
directly or indirectly to said detectable output. Hence, the
selection criterion will be dependent on the detectable output.
For example the predetermined selection criterium may be a
quantitative criterium, such as a quantitative level of
bioluminescence above or below a specific threshold value.
In embodiments of the invention, wherein the detectable output is
fluorescence or the detectable output may be linked to a
fluorescent signal, then the predetermined selection criterion
could be any fluorescence property. For example, the selection
criterion could be intensity of said fluorescence above or below a
predetermined threshold value or emission of light of a specific
wavelength or absorption of light of a specific wavelength or
intensity of emitted light of a specific wavelength above or below
a predetermined threshold value. The selection criterion could also
be based on Fluorescence lifetime and/or fluorescence polarization
The selection criterion could also be a specific localisation of
the fluorescent signal, such as intensity of a fluorescent signal
in a specific cellular compartment above or below a predetermined
threshold value. The selection criterion could also be a
predetermined change in fluorescence lifetime or in fluorescence
polarization. Fluorescence intensity and/or localisation may for
example be determined using image processing and/or image analysis,
a fluorescence microscope, FACS, FABS or fluorescence plate
reader.
In one embodiment of the invention the selection criterion is high
fluorescence intensity. This may for example be the case, when the
cellular response is activation of a signal transduction pathway
and the reporter system comprises a gene encoding a fluorescent
protein, where activation of the signal transduction pathway leads
to increased expression of said gene. This may also be the case
when the cellular response is establishment of interaction between
two cellular proteins, wherein the reporter system is a FRET or
BRET reporter system. After release of a proportion of the library
members to be tested, resin beads may be selected using a method
comprising the steps of: 1. Determining the fluorescence intensity
of positive control resin beads and setting this fluorescence
intensity to 100% 2. Determining the fluorescence intensity of
negative control resin beads and setting this fluorescence
intensity to 0% 3. Selecting resin beads having a fluorescence
intensity corresponding to at least 5%, preferably at least 10%,
more preferably at least 20%, even more preferably at least 30%,
such as at least 40%, for example at least 50%, such as at least
60%, for example at least 70%, such as at least 80%, for example at
least 90%, such as in the range of 5 to 100%, for example in the
range of 10 to 100%, such as in the range of 20 to 100%, for
example in the range of 30 to 100%, such as in the range of 40 to
100%, for example in the range of 50 to 100%.
The positive control may for example be a resin bead (or optionally
several resin beads kept in a separate container or well)
comprising a compound known to influence the cellular response. By
way of example, if the cellular response is activation of an
intracellular signal transduction pathway, then the positive
control may be a resin bead comprising a known compound that
stimulate one or multiple components of said intracellular signal
transduction pathway. The positive control signal is then obtained
after release from the resin of the compound. Alternatively the
positive control may be a resin bead comprising a known ligand of a
receptor activating the signal transduction pathway, for example a
naturally occurring ligand. The negative control may be a resin
bead (or optionally several resin beads kept in a separate
container or well) optionally comprising a cell adhesion compound,
but otherwise comprising no library member or other test
compound.
In another embodiment of the selection criterion is low
fluorescence. This may for example be the case, when the cellular
response is inhibition of a signal transduction pathway and the
reporter system comprises a gene encoding a fluorescent protein,
where an active signal transduction pathway leads to expression of
said gene. This may also be the case when the cellular response is
disruption of interaction between two cellular proteins, wherein
the reporter system is a FRET or BRET reporter system. After
release of a proportion of the library members to be tested, resin
beads may be selected using a method comprising the steps of: 1.
Determining the fluorescence intensity of positive control resin
beads and setting this fluorescence intensity to 0% 2. Determining
the fluorescence intensity of negative control resin beads and
setting this fluorescence intensity to 100% 3. Selecting resin
beads having a fluorescence intensity corresponding to at least 5%,
preferably at least 10%, more preferably at least 20%, even more
preferably at least 30%, such as at least 40%, for example at least
50%, such as at least 60%, for example at least 70%, such as at
least 80%, for example at least 90%, such as in the range of 5 to
100%, for example in the range of 10 to 100%, such as in the range
of 20 to 100%, for example in the range of 30 to 100%, such as in
the range of 40 to 100%, for example in the range of 50 to
100%.
The positive control may for example be a resin bead(s) comprising
a compound known to influence the cellular response. By way of
example, if the cellular response is inhibition of an intracellular
signal transduction pathway, then the positive control may be a
resin bead(s) comprising a compound known to inhibit one or
multiple components of signal transduction pathway. The control
signal is then obtained after release from the resin of the
compound. Alternatively the positive control may be a resin bead
comprising a known antagonist of a receptor known to activate said
signal transduction pathway. If the cellular response is induction
of apoptosis, then the positive control may be a resin bead
comprising a compound known to induce apoptosis The negative
control may be a resin bead optionally comprising a cell adhesion
compound, but otherwise comprising no library member or other test
compound.
One method of selecting resin beads using FABS is illustrated in
FIG. 1A.
In one preferred embodiment selection is performed manually with
the aid of a fluorescence microscope. In this embodiment the
fluorescence intensity or other fluorescence properties are judged
manually.
When the selection criterion is fluorescence intensity of
localisation, the resin beads may also be analysed using a plate
reader or image acquisition. An example of such an analysis is
given in FIG. 1B.
If the selection criterion is localisation, then resin beads are
generally analysed by a fluorescence or imaging microscope. Said
microscope may optionally be equipped with a micromanipulator
capable of picking out single beads. Resin beads are scanned for
cells where the fluorescence signal is located at the desired
intracellular location and these resin beads are selected. The
selection may be manually or it may be automated.
In embodiments of the invention, wherein the detectable output is
light emission or the detectable output may be linked to a light
signal, then the predetermined selection criterion could be any
property of the light. For example the selection criteria could be
light intensity above or below a predetermined threshold value.
Light can be detected for example by the eye, in a microscope, and
if the light is emitted via bioluminescence it can be measured by a
luminometer
In embodiments of the invention, wherein the detectable output is a
radioactive signal or the detectable output may be linked to a
radioactive signal, then the selection criterion could be any
property of said radioactive signal, such as intensity above or
below a predetermined threshold value or localisation of the
radioactive signal.
In embodiments of the invention, wherein the detectable output is a
colour signal or the detectable output may be linked to a colour
signal, then the selection criterion could be any property or said
colour signal. For example the predetermined selection criterion
could be a colour intensity above or below a specific threshold
value or it could be a specific colour. The colour signal could be
detected using any suitable colorimetric method, such as a
spectrophotometer or a microscope.
Resin beads comprising cells meeting at least one selection
criterion, such as any of the selection criteria mentioned herein
above are selected. In certain embodiments of the invention resin
beads comprising cells meeting at least two, for example 2, such as
3, for example 4, such as in the range of 5 to 10, for example of
in the range of 10 to 25 selection criteria are selected.
It is also possible within the present invention to select resin
beads comprising cells meeting one or more predetermined selection
criteria and subsequently to subject said beads to one or more
additional selection rounds, wherein resin beads comprising cells
meeting one or more additional selection criteria are selected.
Resin beads meeting said at least one predetermined selection
criteria may be selected by manually sorting for example with the
aid of a microscope, or for example by sorting by fluorescence or
by colour or by morphology depending on the detectable output and
the selection criterion. Positive beads may be picked directly
under the microscope, such as under a fluorescence microscope for
example manually or with the aid of a micromanipulator. Frequently,
in the range of 100 to 1,000,000, for example in the range of 1000
to 100,000, such as in the range of 5000 to 50,000 resin beads may
be placed on a suitable surface, such as in a dish or on a
coverglass and subsequently examined by microscopy. Alternatively,
the sorting process may be automated with the use of specially
designed, commercially available bead sorters (Union Biometrica,
Sommerville, Mass.) and detecting for example fluorescence
intensity (Meldal, 2002, Biopolymers, 66: 93-100). In general,
resin beads can be sorted at a rate of up to 100 beads per second,
or even faster depending on the equipment used and its reading
capacity. A range of about 5-30 beads per second is generally used
with known instruments. Slower rates may be used to increase
accuracy, however any suitable rate may be used with the present
invention, such as much higher rates. Preferred, is a rate where
only one resin bead passes through the detector at a time. It is
also comprised within the present invention to select resin beads
using a plate reader. In general in the range of 1 to 1000, such as
10 to 500, for example 50 to 100 resin beads are placed in each
well of a multiter plate and analysed. Beads from positive wells
may then be further examined.
In one embodiment of the invention resin beads may be selected by
comparing the detectable output, with the detectable output
generated by control resin beads, for example positive and/or
negative control resin beads. Positive control resin beads are
beads comprising a compound capable of inducing the desired
cellular response, whereas negative control resin beads comprises
no such compound. By way of example, if the cellular response is
activation of an intracellular signal transduction pathway with a
known natural ligand, the positive control resin bead may comprise
said ligand, whereas the negative control resin bead comprises no
compound except optionally a cell adhesion compound.
If the detectable output is a quantifiable signal, then resin beads
may be selected, comprising cells where the detectable output is
higher or lower than the detectable output from cells attached to
the positive or negative control resin bead. By way of example, if
the detectable output is fluorescence intensity, then resin beads
comprising cells displaying a fluorescence intensity which is
higher than the negative control and lower than the positive
control could for example be selected.
Non-limiting examples of methods of selecting resin beads are
illustrates in FIGS. 1 and 2.
Identification of Compound
Once a resin bead has been selected, the compound that is remaining
after partial release on said bead may be identified. Preferably,
only one resin bead is used at a time. Thus if said resin bead only
comprises one library member in one or more copies, then only one
compound is identified at a time.
The process for identification of the library member depends on the
type of library used. For a library of primarily oligomeric
compounds, the library member can be analysed by Mass Spectroscopy
(MS), particularly if the library was synthesized in such a way
that the synthetic history of the compound is captured, for
example, using a capping procedure to generate fragments of the
compound that differ in mass by one building block (see, for
example, Youngquist et al., 1995, J. Am Chem. Soc., 117: 3900-06).
This capping procedure is most efficient when the cap and the
building block are reacted at the same time. The capping agent can
be any class of compound that has at least one functional group in
common with the building block used to generate the oligomer, so
that both the capping agent and the building block can react when
added to the resin in an appropriate ratio. Alternatively, the
capping agent can have two functional groups in common with the
building block where one of the groups in common, such as the group
in the building block that is used for the elongation of the
oligomer, is orthogonally protected. For example, in a synthesis of
a peptide using the Fmoc strategy, the capping agent could be the
same as the building block but with a Boc group protecting the
reactive amine instead of the Fmoc group (see St. Hilaire et al.,
1998, J. Am. Chem. Soc., 120: 13312-13320). In another example, if
the building block is a protected haloamine, the capping agent
could be the corresponding alkylhalide.
Where the library is synthesized by parallel synthesis (a parallel
array), the compound can be identified simply by the knowledge of
what specific reaction components were reacted in a particular
compartment. The structure can be confirmed by cleavage of a small
portion of compound from the solid support and analyzed using
routine analytical chemistry methods such as infrared (IR), nuclear
magnetic resonance (NMR), mass spectroscopy (MS), and elemental
analysis. For a description of various analytical methods useful in
combinatorial chemistry, see: Fitch, 1998-99, Mol. Divers., 4:
39-45; and Analytical Techniques in Combinatorial Chemistry, M. E.
Swartz (Ed), 2000, Marcel Dekker: New York.
In a preferred embodiment however the library has been synthesised
by a split-mix approach where the precise structure of the compound
of a specific bead is unknown. In this embodiment, the library
member can be identified using a variety of methods. The compound
may be cleaved off the resin bead, for example by cleaving the
cleavable linker and then analyzed using IR, MS, or NMR. For NMR
analysis, larger beads containing approximately 5 nmoles of
material are preferably used for the acquisition of 1-dimensional
(1-D) and 2-dimensional (2-D) NMR spectra. Furthermore, these
spectra can be attained using high-resolution MAS NMR (magic angle
spinning nuclear magnetic resonance) techniques.
Alternatively, high resolution-MAS NMR spectra can be acquired
while the ligand is still bound to the solid support, as described
for example, in Gotfredsen et al., 2000, J. Chem. Soc., Perkin
Trans., 1: 1167-71. The compound may also be identified by release
of the compound and fragmentation by MS-MS in MALDI or electrospray
mode.
Frequently, resin beads used for library synthesis contain about
100 to 500 pmoles of material, which is generally insufficient for
direct analysis using NMR techniques. In such situations, the
libraries can be synthesised with special encoding to facilitate
identification of the library member. For a review of encoding
strategies employed in combinatorial chemistry see: Barnes et al.,
2000, Curr. Opin. Chem. Biol., 4: 346-50. Most coding strategies
include the parallel synthesis of the encoding molecule (for
example, DNA. PNA, or peptide) along with the library compounds.
This strategy requires a well-planned, time consuming, orthogonal
protecting group scheme. Furthermore, the encoding molecule itself
can sometimes influence the cell leading to false positives.
Alternatively, the library members can be encoded using
radiofrequency tags or using optical encoding, such as quantum dot
encoding, spherical encoding or distance encoding. These methods
alleviates the problem of false positives stemming from the coding
tags, but is generally only useful for small libraries in a
one-bead-one-compound system due to the sheer bulk of the
radiofrequency tag. Alternatively, single beads can be analyzed in
a non-destructive manner using infrared imaging. This method gives
limited information and while useful for pre-screening, is not
recommended for conclusive structural determination.
In a preferred embodiment of the invention the library member(s)
comprised within selected resin beads are identified using mass
spectrometry (MS). MS can be used alone to identify the library
member. The library member can be cleaved from the resin bead, the
molecular mass determined, and subsequently fragmented into
sub-species to conclusively determine the structure by combination
with knowledge of contained structures in the library. MS-based
methods of compound identification are useful in this invention, as
they require very little material, and can utilise pico- to
femtomole amounts of compound.
After identification of the compound it may be desirable to confirm
the activity of said compounds by further in vitro and/or in vivo
assays. For example, resin beads comprising the identified compound
and optionally an adhesion compound may be synthesized and the
cellular response confirmed. It is also possible to test identified
compounds in in vitro assays in the absence of beads. Cells may for
example be grown directly in a tissue culture dish, flask or
coverglass and the identified compound can be added directly to the
medium of said cells. If several reporter systems are available for
the particular cellular response then preferably several different
reporter assays may be tested in vitro, in order to identify very
useful compounds. By way of example, if the cellular response is
induction of apoptosis, then for example activity of caspases, cell
growth/cell death and/or interaction between Smac and inhibitors of
apoptosis may be tested.
Multiplexing
The methods disclosed by the present invention may also be used in
multiplexing methods.
For example, the methods may be used to identify compounds
modifying at least two cellular responses, such as 2, for example
3, such as 4, for example in the range of 5 to 10, such as in the
range of 10 to 25 cellular responses.
In such methods step c) of the method outlined above (see the
section "Summary of the invention") preferably involves screening
resin beads for beads comprising cells meeting at least two, such
as 2, for example 3, such as 4, for example in the range of 5 to
10, such as in the range of 10 to 25 predetermined selection
criteria, wherein each selection criterion is preferably related to
a different detectable output.
In such a method more than one kind of cell may be attached to each
resin bead and the different cellular responses may be detected in
different kinds of cells. For example, a first cell line comprising
a first reporter system linked to a first cellular response and a
second cell line comprising a second reporter system linked to a
second cellular response and optionally additional cell line(s)
comprising additional reporter system(s) linked to additional
cellular response(s) may all be attached to a single bead. Resin
beads comprising cells meeting selection criteria linked to all the
different reporter systems may then be selected.
Depending on the detectable outputs, said detectable output may be
determined using any of the methods described herein above. In one
preferred embodiment at least two detectable outputs are
fluorescent outputs, preferably of different excitation and/or
emmission. Thus resin beads meeting said at least two selection
criteria may be selected in one step using a FABS with at least 2
channels in both excitation and emmission. Similarly, more than two
different fluorescent properties may be selected for in an suitable
FABS. The at least two detectable outputs may be in the same cell
line or they may be in different cell lines.
Examples of multiplexing methods are illustrated in FIGS. 2A and
2B.
EXAMPLES
Example 1
General Methods for Solid Phase Peptide Synthesis (SPPS)
General for Chemical Synthesis:
All chemicals described, commercially available and used without
further purification. All solvents were HPLC-grade. PEGA-resins
were purchased from VersaMatrix A/S, Copenhagen. Each washing step
lasted 2 min unless otherwise stated. Purifications were performed
on a standard reverse phase HPLC using gradients of
acetonitrile-Water with various amounts of TFA.
Coupling of HMBA Linker to PEGA--Resin:
Dry PEGA-resin was swelled in DCM and washed with DMF (3.times.).
3.0 eq. HMBA, 2.9 eq. TBTU and 3.0 eq. NEM were mixed in
appropriate DMF and allowed to react for 10 min. The mixture was
added to resin and after 2 h the resin was washed with DMF
(6.times.), DCM (6.times.) and lyophilised.
General Procedure for Coupling of Amino Acid to HMBA-Linker:
Dry PEGA-resin with HMBA-linker was swelled in DCM. 3.0 eq.
Fmoc-protected amino acid, 2.25 eq. Melm and 3.0 eq. MSNT were
mixed in appropriate amount of DCM and added to resin. After 1 h
the resin was washed with DCM (3.times.) and the coupling was
repeated as above once. After coupling for 1 h the resin was washed
with DCM (6.times.), DMF (6.times.), DCM (6.times.) and
lyophilised.
General SPPS Coupling Procedure:
The terminal amino acid on the resin was Fmoc-deprotected by
treatment with 20% piperidine in DMF (1.times.2 min+1.times.18 min)
followed by washing with DMF (6.times.). 3.0 eq. Fmoc-protected
amino acid, 2.9 eq. TBTU and 3.0 eq. NEM were mixed in appropriate
amount of DMF and allowed to react for 10 min. The mixture was
added to the resin and after 2 h the resin was washed with DMF
(6.times.).
I. General Side Chain Deprotection Procedure:
Dry PEGA-resin with linker, usually acid stable linker(s) and
peptide or optionally compound was swelled in H.sub.2O and the side
chains was deprotected with 95% TFA (aq) (2.times.15 min). (If Pmc
groups were present cleavage time was 6 h). The resin was washed
with H.sub.2O until washing water had pH=5-7. The resin was then
washed with DMF (6.times.), DCM (6.times.) and lyophilised.
General HMBA Cleavage Procedure:
Dry PEGA-resin with HMBA linker and attached compound was swelled
in water and NaOH (aq.) 0.1 M was added. After 2 h HCl (aq.) 0.1 M
was used for neutralisation and then AcN was added until the
H.sub.2O/AcN ratio was 1:1 by volume. The resin was filtered off
and the liquid was used direct for RP-HPLC or/and Q-TOF MS
analysis.
The above general procedures are used for solid phase peptide
synthesis in the following examples unless otherwise specified.
Example 2a
Screening of Adhesion Peptide Library
Approx. 100 adhesion peptide library beads were mixed with
1.times.10E6 cells (BHK, CHO, U2OS, Hek) in each well of a Falcon
12 well plate using 2 ml growth medium. The adhesion peptide
library was prepared according to the general methods for solid
phase peptide synthesis outlined above. The library consisted of
heptamers of D-amino acids. The peptide library beads were PEGA
beads each coupled to a potential adhesion peptide. The cells and
beads were mixed gently every 15 min for 2 hrs. Supernatant with
non-attached cells were removed and new growth medium added.
Cells/beads were incubating for another 16 hrs. (37.degree. C., 5%
CO.sub.2). Cell adhesive beads were identified using a microscope
with 10.times. objective and positive beads were transferred to a
filter paper (to suck off medium). Peptides were identified by
amino acid sequencing. Examples of useful peptides are given in
table 2.
Example 2b
Identification of an Adhesion Peptide with Low Absorption of
Fluorescent Components from Growth Medium and High Adhesion
Properties
An adhesion D-amino peptide library was synthesized (500.000
members) as described above in Example 2a and screened for low
fluorescence/high adherence properties. This was done in 4 steps:
1) Selection of low fluorescent beads by Fluorescence Activated
Bead Sorting (FABS). The 500.000 member adhesion peptide library
was FABSorted and 150.000 low fluorescent beads were isolated. 2)
Selection of beads with good cell adhesion properties.
The 150.000 low fluorescent beads were incubated with GFP
expressing U2OS cells followed by FABS sorting for high
fluorescence (high cell adhesion). 536 beads were isolated. 3)
Identification and isolation of beads with high Hek293 cell
adherence properties. The 536 beads were cleared for U2OS cells and
incubated with GFP expressing Hek293 cells. 47 beads with high cell
adhesion properties were isolated using a fluorescence microscope.
4) Sequence elucidation and re-synthesis of selected peptides. 22
peptides were sequenced and six of them were re-synthesized. Based
on Structure-Activity of the six peptides, four additional ones
were synthesized. The peptide defined by SEQ ID 35 showed the best
overall performance.
Example 3
Synthesis of Peptides Binding to the ML-IAP BIR Domain
This example describes the preparation of 6 peptides known to
interact with the BIR domain of ML-IAP (livin). The syntheses were
performed according to the "general SPPS coupling Procedure"
above.
Synthesis of H-Ala-Val-Pro-Ile-Ala-Gln-Lys-Ser-Glu-OH (SEQ ID NO.
82), H-Ala-Val-Pro-Phe-Ala-Val-Lys-Ser-Glu-OH (SEQ ID NO. 83), and
H-Ala-Glu-Ala-Val-Pro-Trp-Lys-Ser-Glu-OH (SEQ ID NO. 84)
Coupling 1-3: HMBA modified PEGA 800 beads (600 mg, Versabeads
A-800, 315-500 .mu.m, 0.32 mmol NH2/g) were coupled under standard
MSNT coupling conditions (Example 1) with Fmoc-Glu(O.sup.tBu)OH
followed by standard TBTU coupling of FmocSer(O.sup.tBu)OH and
FmocLys(Boc)OH.
Coupling 4-8: The resin was separated into three 200 mg portions
and three parallel couplings were performed with the respective
amino acids according to the "general SPPS coupling procedure in
Example 1 (Trp, Glu and Gln were side chain protected as the N-Boc,
O-.sup.tBu and N-trityl derivatives respectively). After the last
coupling side chain deprotection was performed according to the
general procedure (Example 1).
The peptides were detached according to the general HMBA cleavage
procedure (Example 1) and the peptides purified with preparative
HPLC.
H-Ala-Val-Pro-Ile-Ala-Gln-Lys-Ser-Glu-OH (SEQ ID NO. 82): Yield
after purification: 30 mg (>98% pure according to HPLC).
MALDI-MS Calcd. (M+H).sup.+ 942.5; Found 942.7. 25 NMR data were in
accordance with the predicted structure.
H-Ala-Val-Pro-Phe-Ala-Val-Lys-Ser-Glu-OH (SEQ ID NO. 83): Yield
after purification: 32 mg (>98% pure according to HPLC).
MALDI-MS. Calcd. (M+H).sup.+ 947.5; Found 948.0. NMR data were in
accordance with the predicted structure.
H-Ala-Glu-Ala-Val-Pro-Trp-Lys-Ser-Glu-OH (SEQ ID NO. 84): Yield
after purification: 38 mg (>98% pure according to HPLC).
MALDI-MS. Calcd. (M+H).sup.+ 1016.5; Found 1016.5. NMR data were in
accordance with the predicted structure.
Synthesis of H-Ala-Val-Pro-Ile-OH (SEQ ID NO. 85)
Synthesis performed according to the general SPPS procedure on 200
mg of HMBA modified PEGA 800 beads. Yield after HMBA cleavage and
purification: 17 mg. (>98% pure according to HPLC). ES-MS.
Calcd. (M+H).sup.+ 399.2. Found 399.2. NMR data were in accordance
with the predicted structure.
Synthesis of H-Ala-Glu-(1-amino-cyclopentanecarboxyl)-Phe-OH (SEQ
ID NO. 86)
Synthesis performed according to the general SPPS procedure on 200
mg of HMBA modified PEGA 800 beads. Yield after HMBA cleavage and
purification: 22 mg. (>98% pure according to HPLC). ES-MS.
Calcd. (M+H).sup.+ 477.2. Found 477.2. NMR data were in accordance
with the predicted structure.
Synthesis of H-Ala-Glu-(azetidine-1-carboxyl)-(homo-Phe)-OH (SEQ ID
NO. 87)
Synthesis performed according to the general SPPS procedure on 200
mg of HMBA modified PEGA 800 beads. Yield after HMBA cleavage and
purification: 21 mg. (>98% pure according to HPLC). ES-MS.
Calcd. (M+H).sup.+ 463.2. Found 463.2. NMR data were in accordance
with the predicted structure.
The structures of the 6 synthesized peptides are given below.
##STR00002## ##STR00003##
Example 4
Synthesis of a 44,000 Member Tetrapeptide Library on Beads with
Fixed Adhesion Peptide
This example describes the preparation of a library of
tetrapeptides (44.000 members) on resin beads containing a "cell
adhesive peptide". This "two compound one bead" (TCOB) library may
be used for in vivo screening of the tetrapeptides for e.g.
antagonists action on the ML-IAP receptor.
FmocLys(Fmoc)/BocVal (.about.1:1) Modification of Pega 1900
Beads
Dry Versabeads A-1900 (315-500 .mu.m, 0.2 mmol NH2/g) (1.20 g) were
dissolved in dry DMF (10 mL) and coupled with FmocLys(Fmoc)OH (0.24
mmol, 1 eq) and Boc-ValOH (0.48 mmol, 2 eq) using the general SPPS
coupling procedure.
Fmoc to Alloc Transformation of Lys Protecting Groups
The beads were Fmoc deprotected using the standard procedure. The
beads were washed several times with DCM (-10.degree. C.) and
finally added DCM (10 mL) with the same temperature. DIPEA (0.9 mL)
was added followed by dropwise addition of AllocCl
(allyloxycarbonylchloride) (0.26 mL). The reaction mixture was kept
under N.sub.2 and stirred at RT for 2 h (Kaiser test was negative).
The resin was washed with DCM (6.times.), DMF (6.times.) and DCM
(6.times.) and lyophilised overnight.
Attachment of Photolabile Linker (PLL) to Val
The dried beads (.about.1.2 g) were Boc deprotected with 30% TFA in
DCM (2+45 min). The resin was washed with DCM (3.times.), 20% pip
in DMF (2.times.) and DMF (6.times.). The photo cleavable linker
(4-[(1-Fmoc-aminoethyl)-2-methoxy-5-nitrophenoxy]butanoic acid)
(187 mg, 0.36 mmol, 3 eq) was dissolved in DMF followed by NEM (100
.mu.L, 0.72 mmol, 6 eq) and TBTU (111 mg, 0.35 mmol, 2.9 eq). After
stirring for 5 minutes the mixture was added to the beads and left
with occasional stirring for 3.5 h. (Kaiser test neg). Wash with
DMF (10.times.), DCM (6.times.) and lyophilised overnight in the
dark.
Preparation of Tetrapeptide Library with 44,000 Members
The beads containing the Fmoc protected photo linker (1.00 g) were
placed in a custom made 20 well synthesizer. Four couplings using
the general SPPS TBTU coupling procedure were performed using a
split and mix protocol. Amino acids (20.times.20.times.10.times.10)
used for couplings are given in Tables 3 to 6. For the 3.sup.rd and
4.sup.th coupling each amino acid is added to 2 wells. Protection
groups are left on the peptides after end couplings.
TABLE-US-00003 TABLE 3 Amino acids used for the 1st coupling.
Member number 1 2 3 4 Structure ##STR00004## ##STR00005##
##STR00006## ##STR00007## Mw Mol. Wt.: 405.42 Mol. Wt.: 405.42 Mol.
Wt: 455.43 Mol. Wt: 412.44 micromol 10 10 10 10 Weight (mg) 4.05
4.05 4.55 4.12 Member number 5 6 7 8 Structure ##STR00008##
##STR00009## ##STR00010## ##STR00011## Mw Mol. Wt.: 412.44 Mol. Wt:
443.51 Mol. Wt.: 393.46 Mol. Wt: 447.48 micromol 10 10 10 10 Weight
(mg) 4.12 4.43 3.93 4.47 Member number 9 10 11 12 Structure
##STR00012## ##STR00013## ##STR00014## ##STR00015## Mw Mol. Wt.:
359.37 Mol. Wt: 351.4 Mol. Wt.: 365.42 Mol. Wt: 610.7 micromol 10
10 10 10 Weight (mg) 3.59 3.51 3.65 6.11 Member number 13 14 15 16
Structure ##STR00016## ##STR00017## ##STR00018## ##STR00019## Mw
Mol. Wt.: 411.45 Mol. Wt.: 387.43 Mol. Wt: 417.45 Mol. Wt.: 421.87
micromol 10 10 10 10 Weight (mg) 4.11 3.87 4.17 4.22 Member number
17 18 19 20 Structure ##STR00020## ##STR00021## ##STR00022##
##STR00023## Mw Mol. Wt.: 413.47 Mol. Wt.: 427.49 Mol. Wt.: 648.77
Mol. Wt: 355.41 micromol 10 10 10 10 Weight (mg) 4.13 4.27 6.49
3.55
TABLE-US-00004 TABLE 4 Amino acids used for the 2nd coupling.
Member number 1 2 3 4 Structure ##STR00024## ##STR00025##
##STR00026## ##STR00027## Mw Mol. Wt.: 337.37 Mol Wt.: 337.37
Mol.Wt.: 355.41 Mol. Wt.: 323.34 micromol 10 10 10 10 Weight (mg)
3.37 3.37 3.55 3.23 Member number 5 6 7 8 Structure ##STR00028##
##STR00029## ##STR00030## ##STR00031## Mw Mol. Wt.:351.4 Mol. Wt.:
365.42 Mol. Wt.: 351.4 Mol. Wt.: 413.47 micromol 10 10 10 10 Weight
(mg) 3.51 3.65 3.51 4.13 Member number 9 10 11 12 Structure
##STR00032## ##STR00033## ##STR00034## ##STR00035## Mw Mol Wt.:
405.42 Mol. Wt.: 387.43 Mol. Wt.: 393.46 Mol. Wt.: 610.7 micromol
10 10 10 10 Weight (mg) 4.05 3.87 3.93 6.11 Member number 13 14 15
16 Structure ##STR00036## ##STR00037## ##STR00038## ##STR00039## Mw
Mol. Wt.: 339.39 Mol. Wt.: 353.41 Mol. Wt.: 411.45 Mol. Wt.: 477.49
micromol 10 10 10 10 Weight (mg) 3.39 3.53 4.11 4.27 Member number
17 18 19 20 Structure ##STR00040## ##STR00041## ##STR00042##
##STR00043## Mw Mol. Wt.: 648.77 Mol. Wt.: 397.46 Mol. Wt.: 477.51
Mol. Wt.: 311.33 micromol 10 10 10 10 Weight (mg) 6.48 3.97 4.77
3.11
TABLE-US-00005 TABLE 5 Amino acids used for the 3rd coupling.
Member number 1 2 3 4 Structure ##STR00044## ##STR00045##
##STR00046## ##STR00047## Mw Mol. Wt.: 339.39 Mol. Wt.: 353.41 Mol.
Wt.: 353.41 Mol. Wt.: 397.46 micromol 10 10 10 10 Weight (mg) 3.39
3.53 3.53 3.97 Member number 5 6 7 8 Structure ##STR00048##
##STR00049## ##STR00050## ##STR00051## Mw Mol. Wt.: 411.45 Mol.
Wt.: 596.67 Mol. Wt.: 425.47 Mol. Wt.: 610.7 micromol 10 10 10 10
Weight (mg) 4.11 5.97 4.25 6.11 Member number 9 10 11 12 Structure
##STR00052## ##STR00053## MW Mol. Wt.: 477.51 Mol. Wt.: 440.49
micromol 10 10 Weight (mg) 4.78 4.40
TABLE-US-00006 TABLE 6: Amino acids used for the 4th coupling.
Member number 1 2 3 4 Structure ##STR00054## ##STR00055##
##STR00056## ##STR00057## Mw Mol. Wt.: 175.18 Mol. Wt.: 189.21 Mol.
Wt.: 217.26 Mol. Wt.: 261.31 micromol 10 10 10 10 Weight (mg) 3.50
3.78 4.35 5.22 Member number 5 6 7 8 Structure ##STR00058##
##STR00059## ##STR00060## ##STR00061## Mw Mol. Wt.: 275.34 Mol.
Wt.: 203.24 Mol. Wt.: 485.66 micromol 10 10 10 10 Weight (mg) 5.51
4.02 4.06 9.71 Member number 9 10 11 12 Structure ##STR00062##
##STR00063## Mw Mol. Wt.: 215.25 Mol. Wt.: 535.7 micromol 10 10
Weight (mg) 4.30 10.71
Attachment of "adhesion peptide":
Adhesion peptides were synthesized on the alloc-protected lysine.
One batch of the library was attached adhesion peptide A, and a
second batch with adhesion peptide B.
Adhesion peptide A:
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-lle-D
-Arg(Pmc)-D-Gln(Trt)-Gly-)
Alloc deprotection of lysine residues: The library beads were
treated under N.sub.2 with 3 eq Pd(PPh.sub.3).sub.4 in 10 mL
degassed CHCl.sub.3 containing 5% HOAc and 2.5% NEM for 2h at RT.
The resin was washed with CHCl.sub.3 (6x), DMF containing 0.5%
sodium diethyl-dithiocarbamat and 0.5% DIPEA (2x), and DMF
(10x).
Coupling: The adhesive peptide was synthesized directly (stepwise)
on the library beads using the general SPPS coupling procedure.
Alternative method: The purified peptide
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-L-
-Gly-OH) (SEQ ID NO. 88) (3 eq) was coupled to the lysine NH.sub.2
groups using the general SPPS coupling procedure.
Adhesion peptide B:
(Fmoc-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)- (SEQ ID
NO. 89):
Analogous to synthesis of adhesion peptide A.
Adhesion Peptide B:
(Fmoc-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)- (SEQ ID
NO. 36)
Analogous to synthesis of adhesion peptide A.
Deprotection of Adhesion and Library Peptides
Final deprotection of protecting groups was performed according to
the general procedure described above yielding the TCOB library
ready for testing
Example 5a
Preparation of TAT Appended "Tetrapeptide" Library
The peptide library of example 4 is modified to include a TAT
sequence appended to the C-terminal end of the peptide library in
order to enhance the cell permeation properties of the
tetrapeptides.
Synthesis of TAT sequence (GGYGRKKRRQRRR; SEQ ID NO. 90) on Val
residues.
Method A: Beads (1.00 g) containing FmocPLL-Val and AllocLys(Alloc)
(as prepared in example 3) are subjected to stepwise SPPS using the
general TBTU protocol giving the sequence FmocGGYGRKKRRQRRR (SEQ ID
NO. 90) attached to the photo labile linker (Arg is side chain
protected as Pmc. Gln protected as Trt, Lys protected as Boc, and
Tyr as .sup.tBu).
Method B: Beads (1.00 g) containing FmocPLL-Val and AllocLys(Alloc)
(as prepared in example 3) are coupled to the side chain protected
and purified TAT sequence FmocG-GYGRKKRRQRRR (SEQ ID NO. 90) using
the general SPPS coupling procedure. Protection of side chains as
in method A.
Formation of TAT Appended Tetrapeptide Library
Using the FmocTAT-Val derivatized beads a tetrapeptide library is
build according to the description in Example 3. The resulting
library is derivatized with the cell adhesive peptide and
followingly all protection groups are removed as described in
example 4.
Example 5b
Preparation of Tetrapeptide Library with 44,000 Members with Base
Labile Adhesion Peptide
In certain preferred embodiments it might be advantageous to be
able to clear selected beads from both cells and adhesion peptide
before either analysis of active compound or further processing.
This example describes the synthesis of the above described library
on beads where the adhesion peptide is linked to the bead via an
HMBA linker.
Synthesis of FmocGly/AllocGly (Ratio .about.1:1) Modified PEGA 1900
Beads
PEGA 1900 beads (300-500 .mu.m, 0.24 mmol NH.sub.2/g) (4 g, 0.96
mmol.about.1 eq)) were treated with a 1:1 TBTU coupling mixture of
FmocGlyOH (2 eq) and AllocGlyOH (2 eq), NEM (17 eq) and TBTU (3.8
eq). Coupling time 3.5 h. Beads washed 10.times. with DMF.
Coupling of Holmes Photolinker to the "Core" Fmoc-Glycine
FmocGly/AllocGly beads were standard Fmoc deprotected with 20%
piperidine in DMF leaving the Alloc glycine untouched. Then
standard TBTU coupling of the Holmes photolinker
(4-(1-aminoethyl-2-methoxy-5-nitrophenoxy)butanoic acid) to the
deprotected glycine (1 eq=0.48 mmol). After coupling, the beads
were washed with DMF and DCM and lyophilized.
Tetrapeptide Library Synthesis on the Photo Linker
Performed analogous to example 4
Alloc Deprotection of Second "Core" Glycine:
The beads from the 20 wells were all combined in a 50 mL syringe
and washed with CHCl.sub.3 (5.times.) and with Ar-degassed
CHCl.sub.3 containing 5% HOAc and 2.5% NEM (5.times.). A solution
of Pd(PPh.sub.3).sub.4 (3 eq, 0.72 mmol) in Ar-degassed CHCl.sub.3
containing 5% HOAc and 2.5% NEM (10 mL) was added to the beads and
after a few minutes bobbling with Ar the syringe was sealed with
parafilm and left for 2 h. The beads were washed with CHCl.sub.3
(10.times.), DMF (10.times.), MeOH (10.times.), DMF (20% pip)
(2.times.), DMF/10.times.). Kaiser test was positive.
Attachment of HMBA Linker
According to standard procedure above.
MSNT Coupling of FmocGlyOH
According to standard procedure above.
TBTU Coupling of FmocLys(Fmoc)OH
According to standard procedure above.
Adhesion Peptide Synthesis
After deprotection of the lysine Fmoc groups performed analogous to
example 4.
Example 5c
Formation of a "Tetrapeptide" Library Linked Via an Internal Amide
Nitrogen
Synthesis of FmocGly/AllocGly (Ratio .about.1:1) Modified PEGA 1900
Beads
PEGA 1900 beads (300-500 .mu.m, 0.24 mmol NH.sub.2/g) (4 g, 0.96
mmol.about.1 eq)) were treated with a 1:1 TBTU coupling mixture of
FmocGlyOH (2 eq) and AllocGlyOH (2 eq), NEM (17 eq) and TBTU (3.8
eq). Coupling time was 3.5 h. Beads were washed 10.times. with
DMF.
Coupling of Aldehyde Photolinker to the "Core" Fmoc-Glycine.
FmocGly/AllocGly beads were standard Fmoc deprotected with 20%
piperidine in DMF leaving the Alloc glycine untouched. Then
standard TBTU coupling of the aldehyde photolinker
(4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoic acid) to the
deprotected glycine (1 eq=0.48 mmol). After coupling, the beads
were washed with DMF and DCM and lyophilized.
TABLE-US-00007 TABLE 7 5 Phenylethylamines used for reductive
amination of aldehyde linker 1 2 3 4 5 ##STR00064## ##STR00065##
##STR00066## ##STR00067## ##STR00068## 121.18 g/mol 139.17 g/mol
139.17 g/mol 155.63 g/mol 151.21 g/mol d: 0.965 d: 1.061 d: 1.066
d: 1.119 d: 1.033
TABLE-US-00008 TABLE 8 Compounds used for BTC coupling Well number
1 2 3 4 Structure ##STR00069## ##STR00070## ##STR00071##
##STR00072## Mw (g/mol) 452.50 452.50 452.50 452.50 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 16.3 16.3 16.3 16.3 Well number 5
6 7 8 Structure ##STR00073## ##STR00074## ##STR00075## ##STR00076##
Mw (g/mol) 452.50 452.50 452.50 452.50 Mol .times. 10.sup.6 36 36
36 36 Weight (mg) 16.3 16.3 16.3 16.3 Well number 9 10 11 12
Structure ##STR00077## ##STR00078## ##STR00079## ##STR00080## Mw
(g/mol) 452.50 452.50 337.67 337.67 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 16.3 16.3 12.1 12.1 Well number 13 14 15 16
Structure ##STR00081## ##STR00082## ##STR00083## ##STR00084## Mw
(g/mol) 323.34 351.40 399.44 351.40 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 11.6 12.7 14.4 12.7 Well number 17 18 19 20
Structure ##STR00085## ##STR00086## ##STR00087## ##STR00088## Mw
(g/mol) 427.49 413.47 379.46 565.72 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 15.4 14.9 13.7 20.4
TABLE-US-00009 TABLE 9 Compounds used for acylation in wells 1-10.
1 2 3 4 5 ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## Mw: 132.59 Mw: 130.53 Mw: 154.60 Mw: 158.56 Mw: 146.62
d: 1.091 d: 1.324 d: 1.167 d: 1.342 d: 1.096 6 7 8 9 10
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
Mw: 120.58 Mw: 134.61 Mw: 106.55 Mw: 104.54 Mw: 78.50 d: 0.980 d:
0.969 d: 1.017 d: 1.151 d: 1.104
TABLE-US-00010 TABLE 10 Compounds used for TBTU coupling Member
number 1 2 3 4 Structure ##STR00099## ##STR00100## ##STR00101##
##STR00102## Mw (g/mol) 353.41 379.46 393.48 411.45 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 12.7 13.7 14.1 14.8 Member number
5 6 7 8 Structure ##STR00103## ##STR00104## ##STR00105##
##STR00106## Mw (g/mol) 405.42 387.43 397.46 351.40 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 14.6 13.9 14.3 12.6 Member number
9 10 11 12 Structure ##STR00107## ##STR00108## ##STR00109##
##STR00110## Mw (g/mol) 353.41 477.51 425.47 596.67 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 12.7 17.2 15.3 21.5 Member number
13 14 15 16 Structure ##STR00111## ##STR00112## ##STR00113##
##STR00114## Mw (g/mol) 565.42 323.34 610.70 447.49 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 20.4 11.6 22.0 16.1 Member number
17 18 19 20 Structure ##STR00115## ##STR00116## ##STR00117##
##STR00118## Mw (g/mol) 325.36 383.44 393.46 311.33 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 11.7 13.8 14.2 11.2
TABLE-US-00011 TABLE 11 Compounds used for TBTU coupling of amino
acids and acylation with acyl chlorides Member number 1 2 3 4
Structure ##STR00119## ##STR00120## ##STR00121## ##STR00122## Mw
203.24 201.22 189.21 189.21 Mol .times. 10.sup.6 36 36 36 36 Weight
(mg) 7.3 7.2 6.8 6.8 Member number 5 6 7 8 Structure ##STR00123##
##STR00124## ##STR00125## ##STR00126## Mw (g/mol) 203.24 203.24
442.63 442.63 Mol .times. 10.sup.6 36 36 36 36 Weight (mg) 7.3 7.3
15.9 15.9 Member number 9 10 11 12 Structure ##STR00127##
##STR00128## ##STR00129## ##STR00130## Mw (g/mol) 175.18 485.66
231.29 231.29 Mol .times. 10.sup.6 36 36 36 36 Weight (mg) 6.3 17.5
8.3 8.3 Member number 13 14 15 16 Structure ##STR00131##
##STR00132## ##STR00133## ##STR00134## Mw (g/mol) 217.26 215.25
275.34 134.60 Mol .times. 10.sup.6 36 36 36 120 Weight (mg) 7.8 7.7
9.9 16.2 (0.017 mL) Member number 17 18 19 20 Structure
##STR00135## ##STR00136## ##STR00137## ##STR00138## Mw (g/mol)
120.58 104.53 106.55 78.50 Mol .times. 10.sup.6 120 120 120 120
Weight (mg) 14.4 (0.015 mL) 12.6 (0.011 mL) 12.8 (0.013 mL) 9.4
(0.009 mL)
Library Synthesis: Step 1, Reductive Amination of Photo Linker
Aldehyde:
5 portions of the above beads (0.8 g dry beads) were placed in 5
syringes of 5 mL.
The beads were swollen in DMF and phenylethylamines (see Table 7)
were coupled to the free aldehyde on the photolinker by reductive
amination as follows: 0.8 g dry beads is 96 .mu.mols.about.1 eq
were pre-treated with the reaction solvent (DMF/HOAc/TEOF/EtOH,
1:1:1:1). Then to each syringe was added one of the five
phenylethylamines (20 eq) dissolved in the reaction solvent (400
.mu.L), and the same solvent was added so the beads were covered.
After 0.5 h NaBH.sub.3CN (20 eq) was added and the beads stirred
cautiously until all dissolved. After 1 h another portion of
NaBH.sub.3CN (20 eq) was added and the mixture left for additional
2 h. The beads were washed with DMF (10.times.), DCM (10.times.),
MeOH (10% HOAc) (2.times.), MeOH (10.times.), DMF (20% pip)
(2.times.), and DMF (10.times.), DCM (10.times.). The 5 samples
were lyophilized over night.
Step 2, Mix and Split of Phenylethyl Amines:
0.4 g of each of the 5 bead samples from above were swelled in DMF,
mixed and transferred to a custom made 20-well library synthesizer
such that each well contained approximate equal amounts of
beads.
Step 3, BTC Coupling of First Amino Acid:
Total amount of beads=2.0 g.about.0.24 mmol gives 12 .mu.mol
NH/well .about.1 eq.
20 BTC-couplings from 11 different amino acids as shown in Table 8
was made as follows: Beads were pre-treated with a 1:1 vol %
THF/DIPEA solution for 5 minutes and drained. Of each amino acid in
Table 2, 3 eq (36 .mu.mol) was dissolved in dry THF (200 .mu.L) and
BTC (1.67 eq) added as 200 .mu.L of a freshly made stock solution
in dry THF. Then 2,4,6-collidine (14 eq), as 200 .mu.L of a freshly
made stock solution in dry THF, was added and the resulting 20
suspensions left for 5 minutes. Each suspension was added to the
respective well and after short mixing the synthesizer was sealed
and left over night with gentle shaking. Next morning the beads
were washed with THF (10.times.) and DMF (10.times.) without mixing
the wells.
Step 4, Acylation of Well 1-10:
Beads in well 1-10 were washed with DCM (10.times.) and Boc
deprotected with 30% TFA in DCM followed by wash with DCM
(10.times.), DCM (5% DIPEA), DMF, and DCM (10.times.). From each of
the 10 acyl chlorides in Table 9 was made a solution of 10 eq acyl
chloride (120 .mu.mol) and DIPEA (20 eq) in dry DCM (400 .mu.L)
containing catalytic amounts of DMAP. The resulting solutions were
added to well 1-10 and left with gentle shaking for 1 h. The
reaction was repeated. After end reactions the beads were washed
with DCM (10.times.), and DMF (10.times.).
Step 5, Removal of Fmoc Protection Groups:
Well 1-20 were standard Fmoc deprotected, followed by wash with DMF
(10.times.).
Step 6, Mix and Split of the 20 Wells
The content of the 20 wells was thoroughly mixed and re-distributed
equally into the wells.
Step 7, Coupling of Second Amino Acid (20 Amino Acids):
To each well was coupled an amino acid according to Table 10 by a
standard TBTU coupling. Coupling time 5 h. After end reaction the
beads were washed with DMF (10.times.).
Step 8, Coupling of Third Amino Acid/Acyl Chloride (15 Amino
Acids-5 Acyl Chlorides):
The beads in all wells were standard Fmoc deprotected and for well
1-15 standard TBTU coupled with the Boc-protected amino acids in
Table 11. For wells 16-20 the beads were first washed with DCM
(10.times.) and the N-terminal amines acylated with the five acyl
chlorides listed in Table 11 (entry 16-20) analogous to step 4.
After couplings all wells were washed with DMF (10.times.).
Step 9, Alloc Deprotection of Second "Core" Glycine:
The beads from the 20 wells were all combined in a 50 mL syringe
and washed with CHCl.sub.3 (5.times.) and with Ar-degassed
CHCl.sub.3 containing 5% HOAc and 2.5% NEM (5.times.). A solution
of Pd(PPh.sub.3).sub.4 (3 eq, 0.72 mmol) in Ar-degassed CHCl.sub.3
containing 5% HOAc and 2.5% NEM (10 mL) was added to the beads and
after bobbling a few minutes with Ar the syringe was sealed with
parafilm and left for 2 h. The beads were washed with CHCl.sub.3
(10.times.), DMF (10.times.), DMF (5% DIPEA, 5% sodium
diethyldithiocarbamate) (5.times.), MeOH (10.times.), DMF DMF
(20.times.). Kaiser test was positive.
Step 10, Attachment of HMBA Linker:
According to the standard procedure above.
Step 11, MSNT Coupling of FmocGlyOH:
According to the standard procedure above.
Step 12, TBTU Coupling of FmocLys(Fmoc)OH
According to the standard procedure above.
Step 13, Attachment of Adhesion Peptides
Adhesion peptides were synthesized on the Fmoc-protected lysine
such that the final beads had two adhesion peptides pr. library
molecule. One batch of the library was attached adhesion peptide A,
and a second batch with adhesion peptide B.
Adhesion peptide A:
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-G-
ly- (SEQ ID NO. 88):
Fmoc deprotection of lysine residues: According to the standard
procedure.
Coupling: The adhesive peptide was synthesized directly (stepwise)
on the library beads using the general SPPS coupling procedure.
Alternative method: The purified peptide
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)D-Ile-D-Arg(Pmc)-D-Gln(Trt)-L--
Gly-OH) (SEQ ID NO. 88) (3 eq) was coupled to the lysine NH.sub.2
groups using the general SPPS coupling procedure.
Adhesion peptide B:
(Fmoc-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-: (SEQ ID:
35)
Analogous to synthesis of adhesion peptide A.
Deprotection of Adhesion and Library Peptides
Final deprotection of protecting groups was performed according to
the general procedure described above yielding the TCOB libraries
ready for testing.
Example 6
Partial Release of Library Compounds from Beads
The library beads of Examples 4 and 5 are swelled in the assay
medium and illuminated with an OMNILUX E-40 (400 W UV lamp, 365 nm,
#89514005, Steinigke Showtechnic GmbH, Germany) for 15 min. The
beads could be placed in any environment (for example plates, wells
etc. used for the assays). This treatment releases appropriate
amounts of active peptide for assay use and the beads withholds
enough active peptide for subsequent identification.
Example 7
Clearance of Cells and Adhesion Peptide
A portion of the library beads with cells attached were drained
from medium and treated with a 0.1 M NaOH solution for 1 h. Beads
were gently spinned down and the supernatant removed. The beads
were washed with water (10.times.) to ensure complete removal of
cells and proteins.
Example 8
Functional assay for identification of compounds that stimulate a
Gs coupled signal transduction pathway (Cre-GFP reporter assay
detected with a Fluorescence Activated Bead Sorter)
Cre-GFP:
Cre-GFP is commercially available from clontech (pCre-d2eGFP) The
vector contains three copies of Cre-binding sequence fused to a
TATA-like promoter. The vector is holding a neomycin resistance
gene. A map of the vector is shown in FIG. 3. MC4R:
PCR amplified MC4R encoding DNA is introduced into the gateway
Entry Vector (pENTR) by topoisomarase-mediated ligation. The DNA is
subsequently recombined into Destination Vector pDEST12.2.
(pDEST12.2MC4R)
Cell Line Establishment:
HEK cells are transfected with pDEST1.2.2MC4R using standard
procedure for Fugene6 transfection. Cells are put under G418
selection for 4 weeks to obtain a cell line stably expressing
MC4R.
The U2OS cell line stably expressing the human MC4R (melanocortin4
receptor) is further transfected with Cre-GFP the day before
culturing them on PEGA beads displaying adhesion peptide and
respectively 1) Negative control (PEGA beads with adhesion peptide,
but no library compound), 2) Positive control (PEGA beads with
adhesion peptide but no library compound) and 3) Library compounds.
The three cultures are handled separately in each their culture
flask.
Bead/Cell Preparation:
Cells are trypsinized and mixed with the PEGA beads in growth
medium (DMEM containing 10% FCS, in the proportion 4000 cells/bead
and app. 50 ml growth medium/5000 beads. 1) Positive control: 50 ml
Growth medium+5000 positive control beads+2.times.10E7 cells+10
.mu.M Forskolin. 2) Negative control: 50 ml Growth medium+5000
negative control beads+2.times.10E7 cells. 3) Screening library
(eg. 100.000 compounds): 1000 ml Growth medium+100.000 library
beads+4.times.10E8 cells was illuminated with an OMNILUX E-40 UV
light (365 nm) for 30 min for obtaining a partial release of the
library compounds (as described in example 6) The three culture
flasks are placed on a Magnetic stirring platform (Techne) designed
for cell culture in suspension and incubated at 37.degree., 5% CO2
for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10
min pause.
Beads, now covered with cells, are allowed to sediment for 10 min
(no centrifugation needed) and the growth medium is removed using a
50 ml pipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently
and left for 15 min. Beads are washed w. 10 ml PBS/5000
beads.times.3 by allowing sedimentation for 10 min between each
wash. Cells are now preserved and fixed to the beads
Bead Sorting:
A Fluorescence Activated Bead Sorter (FABS) equipped with a
multiline Argon laser 488 nm excitation line and 500-650 nm
emission filter and sorting capability into 96 well plate is used
to identify and isolate positive hit beads.
The FABS is calibrated to identify and isolate positive hit beads
(increased GFP fluorescence) by determining the dynamic range of
the assay using positive control beads prepared as described above
as Smax (maximum response) and negative control beads comprising
only cell adhesion peptide as 5 min (minimum response). A cut off
at 30% response compared to negative control beads is set as
threshold for a positive hit bead.
Positive hits are separated into each their well of a 96 well plate
and are hereafter ready for compound elucidation, re-synthesis and
re-test as well as test for effects in other assays.
Example 9
Functional Assay for Identification of Compounds that Inhibit a Gq
Coupled Receptor (Muscarinic M1) Signal Transduction Pathway (Ca++
Mobilization using Fluo-4)
Ca++ Antagonist Assay:
This assay is designed to identify muscarinic M1 antagonist
compounds. The read out is changes in intracellular Ca++ conc.
detected using the Fluo-4 probe from Molecular probes (see
description elsewhere). Positive hits are compounds that inhibit
Carbacol (muscarinic M1 agonist) induced increase in intracellular
Ca++.
U2OS cells are transfected with Muscarinic M1 receptor using
standard procedure for Fugene6 transfection. Cells are put under
zeocin selection for 4 weeks to obtain a cell line stably
expressing the Muscarinic M1 receptor.
U2OS cells expressing the Muscarinic M1 receptor are cultured on
PEGA beads displaying adhesion peptide and respectively 1) Negative
control (Beads comprising only cell adhesion compound), 2) Positive
control (beads comprising cell adhesion compound and Atropine) and
3) Library compounds. The three cultures are handled separately in
each their culture flask.
Bead/Cell Preparation:
Cells are trypsinized and mixed with beads in DMEM containing 10%
FCS in the proportion 4000 cells/bead and app. 50 ml growth
medium/5000 beads. 1) Positive control: 50 ml Growth medium+5000
positive control beads+2.times.10E7 cells. 2) Negative control: 50
ml Growth medium+5000 negative control beads+2.times.10E7 cells. 3)
Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+100.000 library beads+4.times.10E8 cells was illuminated
with an OMNILUX E-40 UV light (365 nm) for 30 min for obtaining a
partial release of the library compounds (as described in example
6)
The three culture flasks are placed on a Magnetic stirring platform
(Techne) designed for cell culture in suspension and incubated at
37.degree., 5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3
min stirring, 10 min pause.
Measurement of changes in the cytoplasmic free calcium
concentration [Ca.sup.2+].sub.i Beads, now covered with cells, are
allowed to sediment for 10 min (no centrifugation needed) and the
growth medium is removed using a 50 ml pipette. 10 ml Krebs Ringer
buffer (KRW; KrebsRingerWollheim, pH 7.4: NaCl 0.14 M, KCL 3.6 mM,
NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4, 7H.sub.2O 0.5 mM,
NaHCO.sub.3, 2H.sub.2O 1.5 mM, D-Glucose 6 mM, CaCl.sub.2 1.5 mM,
HEPES 10 mM) added 1 uM Fluo-4 (Molecular Probes F-14201)+0.02%
Pluronic (Molecular Probes F-127) per 5000 beads is added, mixed
gently and cells/beads are incubated at 37.degree. C. for 30 min.
Beads are hereafter washed w. 10 ml KRW/5000 beads.times.3 by
allowing sedimentation for 10 min between each wash. The Fluo-4
loaded cells are now ready for detection of changes in
[Ca.sup.2+].sub.i.
The fluorescence is monitored in either a Fluorescence Activated
Bead Sorter (FABS) (COPAS from Union Biometrica, US) that is
equipped with multiple laser excitation lines (476 nm, 483 nm, 488
nm, 496 nm, 514 nm, 520 nm, 568 nm, 647 nm, 676 nm) or a
fluorescence plate-reader (Polarstar Optima from BMG Labtech,
Germany) that is equipped with a flash Xenon blitz lamp. Fluo4
fluorescence is detected in FABS by exciting with 488 nm and
collecting the emitted light on to a PMT through a 530.+-.30 nm
emission filter, and on the plate-reader the cells were excited
through a 490.+-.5 nm excitation filter and the emission collected
through a 510.+-.5 nm emission filter. For calculation of the exact
[Ca.sup.2+].sub.i the fluorescence intensity was converted to
[Ca.sup.2+].sub.i by using the equation
[Ca.sup.2+].sub.i=K.sub.D[(F-F.sub.min)/(F.sub.max-F)] where the
dissociation constant K.sub.D is 345 nM, F is fluorescence
intensity, F.sub.min is total fluorescence in the absence of
Ca.sup.2+ and F.sub.max is total fluorescence when Fluo3 is
saturated with Ca.sup.2+. To obtain F.sub.min the cells were
pre-incubated in a calcium low buffer (pH 7.4: NaCl 0.14 M, KCL 3.6
mM, NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4, 7H.sub.2O 0.5
mM, NaHCO.sub.3, 2H.sub.2O 1.5 mM, D-Glucose 6 mM, EGTA 1.5 mM,
HEPES 10 mM) and was challenged with 1 uM ionomycin immediately
before the fluorescence detection. Similarly F.sub.max was obtained
by suspending the cells in a calcium saturated buffer (pH 7.4: NaCl
0.14 M, KCL 3.6 mM, NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4,
7H.sub.2O 0.5 mM, NaHCO.sub.3, 2H.sub.2O 1.5 mM, D-Glucose 6 mM,
CaCl.sub.2 1.5 mM, HEPES 10 mM) and challenged with 1 .mu.M
ionomycin immediately before detection.
In several of our screening assay we do not use exact ion
[Ca.sup.2+].sub.i, but express the response of screening compounds
as relative to control compounds (see below).
Bead Sorting for Fluo-4 Signal:
A Fluorescence Activated Bead Sorter (FABS) equipped with 488 nm
laser excitation line and 528-572 nm emission filter and injection
capability is used to identify and isolate positive hit beads
(=inhibition of Carbachol induced Ca++ response=decreased
fluorescence compared to negative control).
The FABS is calibrated to identify and isolate positive hit beads
by determining the dynamic range of the assay using positive
control beads as Smax (maximum inhibition=minimal fluorescence) and
negative control beads as Smin (minimum inhibition=maximal
fluorescence). Carbacol 1 uM is injected into the flow stream
resulting in an increase in fluorescence for negative control beads
and an unchanged or minor increase in fluorescence for positive
control beads. A cut off at 30% inhibition compared to negative
control beads is set as threshold for a positive hit bead.
Positive hits are separated into each their well of a 96 well plate
and are hereafter ready for compound elucidation, re-synthesis and
re-test as well as test for effects in other assays.
Example 10
Functional assay for identification of compounds that stimulate a
Gs coupled signal transduction pathway (Cre-GFP reporter assay
detected with fluorescence plate reader or fluorescence imaging
equipment)
Cre-GFP:
Cre-GFP is commercially available from clontech (pCre-d2eGFP) The
vector contains three copies of Cre-binding sequence fused to a
TATA-like promoter. The vector is holding a neomycin resistance
gene. A map of the vector is shown in FIG. 3.
MC4R:
PCR amplified MC4R encoding DNA is introduced into the gateway
Entry Vector (pENTR) by topoisomarase-mediated ligation. The DNA
was subsequently recombined into Destination Vector pDEST12.2.
(pDEST12.2MC4R)
Cell Line Establishment:
U2OS cells are transfected with pDEST1.2.2MC4R using standard
procedure for Fugene6 transfection. Cells were put under G418
selection for 4 weeks to obtain a cell line stably expressing the
MC4R.
The U2OS cell line stably expressing the human MC4R (melanocortin4
receptor) is further transfected with Cre-GFP the day before
culturing them on PEGA beads displaying adhesion peptide and
respectively 1) Negative control (PEGA beads with adhesion peptide,
but no library compound), 2) Positive control (PEGA beads of
example 2) and 3) Library compounds. The three cultures are handled
separately in each their culture flask.
Bead/Cell Preparation:
Cells are trypsinized and mixed with the PEGA beads in growth
medium (DMEM containing 10% FCS, in the proportion 4000 cells/bead
and app. 50 ml growth medium/5000 beads. 1) Positive control: 50 ml
Growth medium+5000 positive control beads+2.times.10E7 cells. 2)
Negative control: 50 ml Growth medium+5000 negative control
beads+2.times.10E7 cells. 3) Screening library (eg. 100.000
compounds): 1000 ml Growth medium+100.000 library
beads+4.times.10E8 cells was illuminated with an OMNILUX E-40 UV
light (365 nm) for 30 min for obtaining a partial release of the
library compounds (as described in example 6)
The three culture flasks are placed on a Magnetic stirring platform
(Techne) designed for cell culture in suspension and incubated at
37.degree., 5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3
min stirring, 10 min pause.
Beads, now covered with cells, are allowed to sediment for 10 min
(no centrifugation needed) and the growth medium is removed using a
50 ml pipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently
and left for 15 min. Beads are washed w. 10 ml PBS/5000
beads.times.3 by allowing sedimentation for 10 min between each
wash. Cells are now preserved and fixed to the beads.
Plate Reader Assay:
Control beads as well as library beads are seeded in 384 well black
plates (eg. Nunc) with clear bottom app. 20 beads per well.
Positive and negative controls are placed in dedicated wells in 2
times 4 replicates in each end of the plate. Negative control=20
negative control beads, positive control=one positive control
bead+19 negative control beads. The plates are measured in a
fluorescence plate reader (PolarStar Optima from BMG) using 490+-6
nm excitation filter and 510+-5 nm emission filter. Positive
control wells are used to determine Smax (maximum response)=100%
activity and negative control wells to determine Smin (minimum
response)=0% activity. Beads from wells showing activity >30%
are collected in a tube for re-seeding in a new 384 well plate,
this time having one bead per well. Smin=one negative control bead
and Smax=one positive control bead. Read plates in plate reader and
identify hits beads using same procedure as described above.
Image Acquisition and Analysis:
Control beads as well as library beads are seeded in 384 well black
plates (eg. Nunc) with clear bottom app. 20 beads per well.
Positive and negative control beads are placed in dedicated wells
in 2 times 4 replicates in each end of the plate. Negative
control=20 negative control beads, positive control=one positive
control bead+19 negative control beads. Plates are placed on a
microscope (Zeiss Axiovert 200M) equipped with filters allowing
fluorescence imaging of eGFP (excitation: 490 nm, emission: 510
nm), 10.times. objective and motorized stage. One image is acquired
for each well followed by image analysis (Metamorph) for
identification of hit beads (green). Beads from hit wells are
seeded in a new 384 well plate this time having one bead per well.
Smin=one negative control bead and Smax=one positive control bead.
Image acquisition and analysis described above is repeated and
final hit beads are identified.
Alternatively, approximately 5000 beads are seeded into Lab-Tech
Chambered Coverglass System (#155361; Nalge Nunc International),
imaging acquisition analysis is performed using the fluorescence
equipment described above, and individual beads that display the
required fluorescence properties are isolated using a
micromanipulator system (Eppendorf Injectman NK).
Example 11
Assay for Identification of Protein-Protein Interaction
(ML-IAP:Smac) Modulators
The present assay that is based on the principle: Bioluminescence
Resonance Energy Transfer (BRET.sup.2), commercially available from
Perkin Elmer. One of two interacting proteins is fused to a
bioluminescent donor (luciferase, Rluc) and the other protein is
fused to a fluorescent acceptor (GFP).
Melanoma Inhibitor of Apoptosis Protein (ML-IAP) Probe:
The probe is constructed by PCR amplification of the ML-IAP
(GenBank Accession number: NM.sub.--139317 (alpha form);
NM.sub.--022161 (beta form) (the accession number refer to the
sequence available on 25 May 2005) from human cDNA libraries
(Marathon-Ready cDNA Library of human kidney cell line and a human
fetal brain cell line, Clontech Laboratories, Inc.) using the
primers livin-F1 (5'-CCA GTG TTC CCT CCA TGG GAC CTA A-3') (SEQ ID
71) and livin-R1 (5'-TAA GCC ATC CCC CAC GCC AAG-3') (SEQ ID 72).
The ML-IAP cDNA gene fragments are further modified to contain
restriction sites by PCR amplification using the primers Livin-F3
(5'-GAT AAG CTT CCA GTG TTC CCT CCA TGG GA-3') (SEQ ID 73),
Livin-R3 (5'-TAT GGA TCC AAG GTG CGC ACG CGG CT-3') (SEQ ID 74),
and Livin-F4 (5'-GAG AAT TCT CCT AAA GAC AGT GCC AAG TG-3') (SEQ ID
75) for amplification of full-length ML-IAP. The primers
Livin-BIR-F1 (5'-CAT GGTACC ATG ACA GAG GAG GAA GAG GAG-3') (SEQ ID
76) and Livin-BIR-R1 (5'-GC TGG ATC CGG GTC CCA GGA GCC CAG-3')
(SEQ ID 77) are used for amplification of the BIR domain of ML-IAP.
The restriction enzyme-treated amplificons are ligated in the BRET2
vectors (obtained from Perkin Elmer, Inc.) to produce N- and
C-terminal in-frame fusions to GFP and Rluc. Smac Probe:
The probe is constructed by ligating restriction enzyme treated PCR
amplification products of the cDNA for human Smac (GenBank
Accession NM.sub.--019887 (variant 1); NM.sub.--138929 (variant 3))
in to the BRET2 vectors (obtained from Perkin Elmer, Inc.) to
produce C-terminal in-frame fusions to GFP and Rluc, respectively.
The following primers are used for PCR: Smac-F1 (5'-GCG CTG CAC AAT
GGC GGC TCT-3') (SEQ ID 78), Smac-R1 (5'-GCA CTC ACA GCT CAC AAA
GGC GTC T-3') (SEQ ID 79), Smac-F3 (5'-GAT GGT ACC CGC TGC ACA ATG
GCG GCT CT-3') (SEQ ID 80), and Smac-R3 (5'-CGT GGA TCC TCA CGC AGG
TAG GCC TCC-3') (SEQ ID 81). Cytosolic expression of biologically
Smac may be achieved by constructing an ubiquitin-smac fusion as
described by Allison M. Hunter, Dan Kottachchi, Jennifer Lewis,
Colin S. Duckett, Robert G. Korneluk, and P. Liston. A Novel
Ubiquitin Fusion System Bypasses the Mitochondria and Generates
Biologically Active Smac/DIABLO. J. Biol. Chem. 278 (9):7494-7499,
2003.
Cell Line Establishment:
Cells are co-transfected with the BRET fusions of ML-IAP and Smac
using standard procedure for Fugene6 transfection. Cells are put
under G418 and zeocin selection for 4 weeks to obtain a cell line
stably expressing the two genes.
Selection of Stable Cells:
The HeLa cell line stably expressing both probes are sorted into a
microtitre plate (one cell per well) for high GFP fluorescence
using a Fluorescence Activated Cell Sorter. The sorted cells are
grown for a week, and split into a plate that is optimized for
bio-luminescence. Final cell-clones are selected, after addition of
the luciferase substrate, DeepBlueC (Perkin Elmer), for high
luminescence (detected with a PolarStar, BMG).
Bead/Cell Preparation:
HeLa/mammalian cells are cultured on PEGA beads displaying adhesion
peptide and respectively 1) Negative control (PEGA beads with
adhesion peptide, and a compound with no activity as the library
component), 2) Positive control (PEGA beads with adhesion peptide,
and a control compound, which disrupts the ML-IAP:Smac interaction
e.g. the compound of Example 20 of WO2004/005248, and 3) Library
compounds (PEGA beads with adhesion peptide, and screening
compounds prepared as described in example 5b or 5c). The three
cultures are handled separately in each their culture flask.
Cells are trypsinized and mixed with the PEGA beads in growth
medium (DMEM containing 10% FCS, in the proportion 4000 cells/bead
and app. 50 ml growth medium/5000 beads. Culture flasks are placed
on a Magnetic stirring platform (Techne) designed for cell culture
in suspension and incubated at 37.degree., 5% CO.sub.2 for 16-24
hrs using spinning interval 30 rpm, 3 min stirring, 10 min pause.
Alternatively, the culture flasks are gently rocked a few
times.
Cell-Coated Beads are Treated as Follows:
1) Positive control (low BRET signal): 50 ml Growth medium+approx.
5000 cell-coated control beads. 2) Negative control (high BRET
signal): 50 ml Growth medium+approx. 5000 cell-coated control beads
3) Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+approx. 100.000 cell-coated library beads. The cell-coated
beads are transferred to assay buffer (either KRW (Krebs Ringer
Wollheim) or PBS (phosphate buffered saline) and illuminated with
UV light (365 nm) for 2-20 min for obtaining a partial release of
the library compounds (as described in example 6). The illuminated
beads are further incubated at 37.degree., 5% CO.sub.2 for 15 min.
Beads, now covered with cells, are allowed to sediment for 10 min
(no centrifugation needed) and the growth medium is removed using a
50 ml pipette.
Positive beads may be indentified by analysing individual beads in
a BRET-compatible microplate reader using 410-80 (370-450 nm) and
515-30 (500-530 nm) bandpass filters for dual emission measurements
of Rluc-luminescence and GFP-fluorescence, respectively. The
luciferase substrate, DeepBlueC, is added by an on-board injector
in the microplate reader. The BRET-signal for the protein-protein
interaction is calculated as the ratio of emission at 515 nm and
emission at 410 nm.
Alternatively Positive Beads May be Identified by Bead Sorting:
A Fluorescence Activated Bead Sorter (FABS) equipped with filters
for BRET detection and sorting capability into 96 well plate is
used to identify and isolate positive hit beads.
The luciferase substrate, DeepBlueC is injected into the flow
stream to emit luciferase-mediated luminescence.
The FABS is calibrated to identify and isolate positive hit beads
(decreased BRET signal) by determining the dynamic range of the
assay using positive control beads prepared as described in above
as Smax (maximum response) and negative control beads comprising
only cell adhesion peptide as Smin (minimum response). Positive
hits are separated into each their well of a 96 well plate and are
hereafter ready for compound elucidation, re-synthesis and re-test
as well as test for effects in other assays.
Example 12
Caspase Activity Assay
Inhibitor of apoptosis proteins (IAPs) interact with caspases to
inhibit their activation and thereby cause a repression of
apoptosis. We seek to identify compounds, which can bind to IAP
with a higher affinity than caspase and, in turn, relieve
IAP-mediated caspase-inhibition. A screening assay is established
that measures caspase activation in response to an apoptosis
inducing signal (e.g. staurosporine, STS) and in the presence of
library compounds with putative IAP-binding activity.
The assay is based on measurements of caspase activity in whole
mammalian cells (HeLa/MCF-7/MeWo), preferably HeLa/SK-MeI28
cultured on PEGA beads displaying adhesion peptide and respectively
1) Negative control (PEGA beads with adhesion peptide, and a
compound with no activity as the library component), 2) Positive
control (PEGA beads with adhesion peptide, and a control peptide
that disrupts the ML-IAP:Smac interaction prepared as described in
example 3 and 3) Library compounds (PEGA beads with adhesion
peptide, and screening compounds prepared as described in example
5b or 5c). The three cultures are handled separately in each their
culture flask.
Caspase activity is measured in bead-attached whole cells by
incubating the cells with a fluorogenic caspase substrate with a
quenching leaving group, e.g. DEVD-Rh1 10-C8 or the CellProbe HT
Caspase 3/7 Whole Cell Assay, Beckman Coulter, Inc.). A fluorogenic
counter stain with different emission properties is applied to
correct for cellmass content on the individual beads.
Caspase substrate (DEVD-R-C8) is described in the following
publication: Sui Xiong Cai, Han Zhong Zhang, John Guastella, John
Drewe, Wu Yang, and Eckard Weber. Design and synthesis of Rhodamine
110 derivative and Caspase-3 substrate for enzyme and cell-based
fluorescent assay. Bioorganic & Medicinal Chemistry Letters 11
(1):39-42, 2001 and in the patent: U.S. Pat. No. 6,342,611 B1 (Jan.
29, 2002, Cytovia, Inc. San Diego, Calif.)
Bead/Cell Preparation:
Cells are trypsinized and mixed with the PEGA beads in growth
medium (DMEM containing 10% FCS, in the proportion 4000 cells/bead
and app. 50 ml growth medium/5000 beads. The beads/cells are placed
in three culture flasks and placed on a Magnetic stirring platform
(Techne) designed for cell culture in suspension incubated at
37.degree., 5% CO2 for 16-24 hours using spinning interval 30 rpm,
3 min stirring, 10 min pause.
Cell-Coated Beads are Treated as Follows:
1) Positive control: 50 ml Growth medium+approx. 5000 Positive
control beads. 2) Negative control: 50 ml Growth medium+approx.
5000 Negative control beads. 3) Screening library (eg. 100.000
compounds): 1000 ml Growth medium+approx. 100.000 cell-coated
Library compound beads. Screening Procedure:
The cell-coated beads are transferred to assay buffer (KRW or PBS
and illuminated with UV light (365 nm) for 30 min for obtaining a
partial release of the library compounds (as described in example
6). The three culture flasks are placed on a Magnetic stirring
platform (Techne) designed for cell culture in suspension and
incubated at 37.degree., 5% CO.sub.2 for 3 to 6 hrs.
The beads are then allowed to sediment for 10 min (no
centrifugation needed) and the growth medium is removed using a 50
ml pipette. 1 ml medium containing the fluorogenic caspase
substrate and reagent buffer is added to the beads. The beads are
incubated without stirring at 37.degree., 5% CO.sub.2 for 0.5-5
hours. The beads are then fixed by treatment with 10 ml 99% EtOH
per 5000 beads, gentle mixing whereafter they are left for 15 min.
Beads are washed w. 10 ml PBS/5000 beads.times.3 by allowing
sedimentation for 10 min between each wash. Alternatively, beads
may be fixed with paraformaldehyde, acetone or zinc-based
fixatives. Cells are now preserved and fixed to the beads.
Bead Sorting:
The beads are analysed in a fashion similar to the procedure
described in example 11. The Fluorescence Activated Bead Sorter
(FABS) is equipped with 488 nm excitation filters and 500-550 nm
emission filter for rhodamine-110 measurements and sorting
capability into 96 well plate is used to identify and isolate
positive hit beads.
The FABS is calibrated to identify and isolate positive hit beads
(increased caspase activity) by determining the dynamic range of
the assay using positive control beads with apoptosis induced cells
to determine the Smax (maximum response) and negative control beads
with uninduced cells as Smin (minimum response).
Positive hits are separated into each their well of a 96 well plate
and are hereafter ready for compound elucidation, re-synthesis and
re-test as well as test for effects in other assays.
Microscopy:
An alternative to FABS-based selection of positive hit beads is
epifluorescence microscopy using standard FITC filters (excitation
480.+-.15 nm, emission 535.+-.20 nm) for visualization of rhodamine
110 fluorescence. The microscope is equipped with micromanipulators
for extraction of positive hit beads.
Example 12b
Detection of Caspase Activity Using Proximity Ligation-Based
Assay
Caspase activity is detected with an assay based on the proximity
ligation technology described by S. Fredriksson, M. Gullberg, J.
Jarvius, C. Olsson, K. Pietras, S. M. Gustafsdottir, A. Ostman, and
U. Landegren. Protein detection using proximity-dependent DNA
ligation assays. Nat. Biotechnol. 20 (5):473-477, 2002. This assay
allows direct detection of both active caspases and cleaved caspase
substrates for example PAPR. Furthermore, the assay can be applied
to fixed samples of primary tissue.
We have developed the proximity ligationtechnique to emcompass a
high volume discovery platform that allow identification of primary
hit compounds in a screening system that because of the ability to
conduct the screening in primary cells closely resembles the
conditions observed directly in the patient.
The assay employs two antibodies, an antibody specific for cleaved
caspase-3 and an antibody for recognizing both the pro-enzyme and
the cleaved form of caspase-3. Both antibodies are conjugated with
an oligo nucleotide. Coordinated and proximal binding of the two
antibodies to an activated caspase allows hybridisation and
ligation of DNA probes which can be amplified by rolling circle
amplification using fluorescently labelled oligonucleotides or
molecular beacons. The quantitative incorporation of a fluorescence
label may thus be done like in quantitative real-time PCR.
Detection and selection of beads with a positive fluorescence
signal is conducted either with the
Fluorescence-Activated-Bead-Sorter or a fluorescence microscope or
a plate reader.
Example 13
Identification of Compound
Once a resin bead is selected, the cells may be cleared off the
beads either by extensive washing or in case of HMBA-linked
adhesion peptides by treatment with 0.1 M NaOH followed by washing
(example 7). The library compound comprised within the bead may
then be identified. Selected bead(s) are washed and swelled in a
small drop of pure water and irradiated for 30 min. with an OMNILUX
E-40 (400 W UV lamp, 365 nm, #89514005, Steinigke Showtechnic GmbH,
Germany). The compound is identified with advanced mass
spectrometry combined with single bead and/or nano-scale NMR
techniques. For example advanced MS may be ES MS-MS analysis on a
MicroMass QTOF Global Ultima mass spectrometer (mobile phase 50%
CH.sub.3CN (aq), 0.1 .mu.L/min) employing a linear ramping of the
collision energy. The spectra are analyzed by generating the exact
mass differences between fragment ions and tabulated to provide the
fragmentation pathway and from that the structure of the compound
released from the selected bead is elucidated. Examples of spectra,
mass differences and fragmentation pathways are given in FIGS. 5 to
7.
Abbreviations
HGF: Hepatocyte Growth Factor NGF: Nerve Growth Factor PDGF:
Platelet Derived Growth Factor FGF: Fibroblast Growth Factor EGF:
epidermal Growth Factor GH: Growth hormone TRE: TPA Response
Element SRE: serum response element CRE: cAMP response element AcN:
acetonitril; Boc: tert-butoxycarbonyl; .sup.tBu: tert-butyl; DCM:
dichloromethane; DMF: dimethylformamide; Fmoc:
9-fluorenylmethoxycarbonyl; HMBA: 4-hydroxymethylbenzoic acid;
Q-TOF MS: quadrupole time-of-flight mass spectrometry; MeIm:
N-methyl imidazole; MSNT:
1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-triazole; NEM: N-ethyl
morpholine; PEGA: polyethylene glycol-polydimethyl acrylamide
resin; Pfp: pentafluorophenyl; Pmc:
2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP-HPLC: reversed phase
high pressure liquid chromatography; SPPS: solid phase peptide
synthesis; TBTU: O-(benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate; TCOB: Two-compound-one-bead TFA: trifluoro
acetic acid; Trt: Trityl.
SEQUENCE LISTINGS
1
8117PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 1Ala Arg Ile Arg Ile Gln His1 527PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 2Ala Lys Cys Arg
Trp Cys Met1 537PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 3Ala Lys Ala Arg Cys Lys Ser1 547PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 4Ala Lys Tyr Trp
Ser Tyr Lys1 557PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 5Ala Tyr Tyr Cys Gln Gln Arg1 567PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 6Ala Arg Arg Cys
Phe Arg Asp1 577PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 7Ala Ala Arg His Cys Tyr Tyr1 587PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 8Ala Tyr Tyr Cys
Gln Gln Arg1 597PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 9Ala Asp Leu Lys Arg Pro Met1 5107PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 10Ala Gly Gly
Lys Arg Lys Phe1 5117PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 11Ala Pro Arg Lys Arg Cys Gly1
5127PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 12Ala Thr Arg Arg Val Ala Arg1 5137PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 13Ala Gly Lys
Lys Asn Lys Asn1 5147PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 14Ala Ala Lys Arg Trp Lys Phe1
5157PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 15Ala Arg Trp Pro Tyr Arg Gly1 5167PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 16Ala Leu Tyr
Trp Thr Trp Arg1 5177PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 17Ala Ala Tyr Arg Trp Tyr Arg1
5187PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 18Ala Arg Cys Ile Arg Gly Asp1 5197PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 19Ala Thr Lys
Cys Lys Gly Arg1 5207PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 20Ala Val Tyr Met Arg Asn Ile1
5217PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 21Ala Arg Lys Arg Ile Arg Gln1 5227PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 22Ala Lys Ile
Arg Glu Lys Arg1 5237PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 23Ala Arg Arg Phe Lys Met Tyr1
5244PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 24Arg Arg Phe Lys1254PRTArtificial SequenceRandomly
synthesized cell adhesion peptide 25Arg Arg Ile
Arg1266PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 26Leu Arg His Arg Leu Lys1 5275PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 27Lys Phe Gly
Gln Lys1 5286PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 28Lys Val Tyr Met His Lys1 5296PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 29Ile Arg Tyr
Arg Leu Arg1 5306PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 30Ala Gln Arg Pro Arg Trp1 5316PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 31Trp Tyr Ala
Lys Arg Arg1 5328PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 32Lys Arg Ile Arg Gln Arg Leu Arg1
5337PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 33Lys Arg Ile Arg Gln Arg Lys1 5345PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 34Arg Ile Arg
Gln Arg1 5355PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 35Arg Gln Arg Ile Arg1 5366PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 36Lys Phe Gly
Gln Lys Cys1 5376PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 37Arg Arg Leu Leu Pro Ile1 5386PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 38Pro Phe Arg
Lys Lys Cys1 5396PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 39Tyr Arg Trp Arg Ile Ala1 5406PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 40Arg Ser Lys
Arg Ile Asn1 5416PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 41Arg Ser Ala Lys Arg Cys1 5426PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 42Lys Lys Gln
Phe Trp Phe1 5436PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 43Arg Met Lys Leu His Lys1 5446PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 44Arg His Trp
Gly Arg Ile1 5456PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 45Thr Lys Arg Leu Lys Thr1 5466PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 46Thr Lys Gly
Lys Ala Lys1 5476PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 47Ala Lys Thr Arg His Arg1 5486PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 48Asn Arg Pro
Arg Val Arg1 5496PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 49Val Pro Arg Lys Val Gln1 5506PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 50Lys Met Arg
Tyr Cys Gln1 5516PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 51Ile Arg Lys His Leu Ile1 5526PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 52Pro Arg Arg
Val Val Ile1 5536PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 53Lys Arg Glu Ser Lys Arg1 5546PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 54Ser Arg Lys
Asp Arg Lys1 5556PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 55Arg Cys Lys Lys Leu Ile1 5566PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 56Arg Lys Leu
Arg Val Asn1 5576PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 57Val Arg Thr Val Arg Val1 5586PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 58Arg Ala Phe
Lys Tyr Tyr1 5596PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 59Ile Thr Arg Arg Thr Gln1 5606PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 60Lys Met Pro
Lys Lys Asn1 5616PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 61Lys Pro Lys Met Met Cys1 5626PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 62Lys Lys Met
Arg Phe Trp1 5636PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 63Lys Lys Lys Phe Tyr Tyr1 5646PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 64Lys Ser Asn
Lys Val Arg1 5656PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 65Lys Trp Pro His His Arg1 5666PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 66Arg His Ile
Gln Trp Tyr1 5676PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 67Leu Arg Leu Lys Pro Lys1 5686PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 68Glu Arg Lys
Arg Cys Thr1 5696PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 69Arg Arg Ala Arg Gln Asp1 5706PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 70Arg Glu Lys
Gly Ala Arg1 57125DNAArtificial SequencePrimer 71ccagtgttcc
ctccatggga cctaa 257221DNAArtificial SequencePrimer 72taagccatcc
cccacgccaa g 217329DNAArtificial SequencePrimer 73gataagcttc
cagtgttccc tccatggga 297426DNAArtificial SequencePrimer
74tatggatcca aggtgcgcac gcggct 267529DNAArtificial SequencePrimer
75gagaattctc ctaaagacag tgccaagtg 297630DNAArtificial
SequencePrimer 76catggtacca tgacagagga ggaagaggag
307726DNAArtificial SequencePrimer 77gctggatccg ggtcccagga gcccag
267821DNAArtificial SequencePrimer 78gcgctgcaca atggcggctc t
217925DNAArtificial SequencePrimer 79gcactcacag ctcacaaagg cgtct
258029DNAArtificial SequencePrimer 80gatggtaccc gctgcacaat
ggcggctct 298124DNAArtificial SequencePrimer 81ggatcctcac
gcaggtaggc ctcc 24
* * * * *
References